Skip to main content
NCBI home page
Neuro-Oncology logoLink to Neuro-Oncology
. 2013 Apr;15(Suppl 1):i1–i51. doi: 10.1093/neuonc/not047

Abstracts

PMCID: PMC3635504
Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0003. PRELIMINARY STUDY Rg3-NANOPARTICLES TREAT GLIOBLASTOMA CELLS

Li Bie 1, Yan Ju 1, Zheng Jin 1

Abstract

INTRODUCTION: Ginsenoside Rg3 is a natural active ingredient that is extracted from Korean red ginseng root. It elevates therapeutic effect of radiotherapy and chemotherapy, but the study found that the application of Rg3 is heavily limited by its low bioavailability and poor absorption via oral administration. METHOD: Rg3-loaded PEG-PLGA-NPs (Rg3-NPs) were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the physicochemical characteristics of Rg3-NPs were investigated in our study. We treated primary glioblastoma with 50 µM Rg3-NPs for 48h. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. RESULTS: MTT shows that the growth of cells can be significantly inhibited by Rg3-NPs in a dose-dependence manner. FCM test shows Rg3-NPs can be released from the conjugate nanoparticle and react with the genes in the cell nuclei causing changes in the gene molecules. We also found that cancer cells treated with Rg3-NPs undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes as determined by microarray-analysis and verified by Real-Time PCR. Furthermore, Rg3-NPs induced the expression of apoptotic and anti-migratory proteins p53 in cell lines. CONCLUSIONS: The results of the present study, together with the results of earlier studies show that Rg3-NPs targets genes involved in the progression of the M-phase of the cell cycle. It is associated with several important pathways, which include apoptosis (p53). Rg3-NPs may be a potent cell-cycle regulation drug targeting the M-phase in glioblastoma cell lines.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0004. INDUCTION OF THE ASTROCYTIC LINEAGE PATHWAY SELECTIVELY ENHANCES THE CHEMOTHERAPEUTIC POTENTIAL OF TEMOZOLOMIDE AND CLOMIPRAMINE IN STEM-CELL ENRICHED PAEDIATRIC GLIOBLASTOMA

Laura Donovan 1, Suzanne Birks 1, Laura Grunewald 1, Filip Zmuda 1, Geoffrey Pilkington 1

Abstract

INTRODUCTION: The treatment of glioblastomas remains a considerable therapeutic challenge due to their heterogenous nature and resistance to current treatment regimens. The cancer stem cell (CSC) population is emerging as a critical target for successful chemotherapeutic treatments as this small subset of cells show an increased resistance to radio- and chemo- therapeutic agents. The induction of astrocytic differentiation plus sequential mitochondrial-mediated (Clomipramine) or DNA-alkylating (Temozolomide) therapy is an attractive method which may render such CSCs more susceptible to treatment. METHOD: Using a patient-derived early passage paediatric glioblastoma, expressing unprecedented levels of CD133 under normoxic (5% CO2, 25% O2) and hypoxic conditions (5% CO2, 3% O2) differentiation towards an astrocytic phenotype was induced over 72 hours with dbcAMP (1mM) and theophylline (1mM) before analysing the expression of CD133, Sox2, Olig2, Musashi1, GFAP, STAT3, βIII-tubulin and O4 by flow cytometry and western blot. Assays for viability, apoptosis, invasion and proliferation and live cell imaging for cell migration were conducted downstream of Temozolomide and Clomipramine treatments. RESULTS: Differentiation along the astrocytic lineage not only resulted in arbitration and complexity of morphology but also decreased the average amount of expression as well as the percentage of the cells expressing all stem cell antigens analysed. Significantly, this treated population of cells showed enhanced response to Clomipramine and Temozolomide in terms of apoptotic cell death. CONCLUSIONS: Inducing the astrocytic lineage pathway and subsequently reducing the stem-like phenotype within paediatric glioblastoma significantly enhances the therapeutic potential of mitochondrially-mediated and DNA-alkylating agents in vitro.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0005. DEFINING THE CELL TYPE- AND BRAIN REGION-SPECIFIC DETERMINANTS UNDERLYING KIAA1549:BRAF-INDUCED MURINE GLIOMA IN VITRO AND IN VIVO

Aparna Kaul 1, Yi-Hsien Chen 1, Sonika Dahiya 1, Ryan Emnett 1, Scott Gianino 1, David Gutmann 1

Abstract

INTRODUCTION: Tandem duplications involving the BRAF kinase gene have recently been identified as the most frequent genetic alteration in sporadic pediatric glioma, creating a novel fusion protein (KIAA1549:BRAF; f-BRAF) with increased BRAF and MEK activity. To define the role of f-BRAF in murine gliomagenesis, we demonstrate that f-BRAF regulates proliferation in a cell type- and brain region-specific manner. METHOD: Primary astrocytes and neural stem cells (NSCs) were employed for the in vitro experiments. Cerebellar NSC implantation was performed by stereotactic injection into the cerebella of naïve mice in vivo. A novel genetically-engineered mouse (GEM) strain was developed in which f-BRAF expression is controlled by both Cre recombinase activity and doxycycline treatment. RESULTS: First, f-BRAF expression increased mouse NSC, but not astrocyte, proliferation in vitro. Second, f-BRAF expression increased cerebellar and third ventricle NSC proliferation in vitro, but had no effect on cortical or lateral ventricle NSC growth. Third, f-BRAF-expressing cerebellar NSCs induced low-grade glioma-like lesions when implanted into the cerebellum in vivo. Fourth, while f-BRAF expression leads to increased MEK activation in all cell types examined, f-BRAF-driven NSC proliferation results from tuberin/Rheb-mediated mTOR hyperactivation, leading to S6-kinase-dependent degradation of p27. Lastly, preliminary studies revealed that conditional f-BRAF expression in BLBP+ NSCs led to increased astrocyte number specifically in the cerebellum. CONCLUSIONS: Collectively, these results establish that cell type- and brain region-specific determinants partly explain the spatial predilection for pilocytic astrocytomas to form in the cerebella of young children. Furthermore, we show that mTOR pathway activation represents a key growth regulatory mechanism common to both sporadic and familial low-grade gliomas in children. With the development of two potential preclinical model (explant and GEM) systems, further mechanistic explorations as well as therapeutic drug testing now become possible.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0006. SIMULTANEOUSLY ACTIVATED Shh And Wnt SIGNALING IN NEURAL PRECURSORS IS SUFFICIENT TO INDUCE SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMORS (sPNET)

Julia Pöschl 1, Edoardo Bianchi 1, Marie Bockstaller 1, Philipp Neumann 2, Ulrich Schüller 1

Abstract

INTRODUCTION: Supratentorial primitive neuroectodermal tumors (sPNET) are malignant forebrain tumors typically arising in early childhood. The biology of sPNETs is largely unknown, and targeted therapies do not exist, mainly due to the lack of mouse models that may be used for preclinical studies. Recently, global gene expression patterns identified three different molecular subgroups of sPNETs with one of them displaying a concurrent upregulation of Wnt- and Sonic hedgehog (Shh) target genes, also referred to as Embryonal tumor with multilayered rosettes (ETMR). METHOD: In order to generate a genetically tailored mouse model, we used the Cre-loxP system and conditionally activated both Shh and Wnt signaling in hGFAP-positive precursor cells of the CNS. Brains from such mice were thoroughly investigated by standard histology, immunohistochemistry and global gene expression analysis. RESULTS: Constitutive activation of both Shh and Wnt signaling in neural CNS precursor cells resulted in the formation of forebrain tumors that displayed remarkable similarities to human sPNETs with respect to localization, morphology and global gene expression patterns. The investigation of early embryonic stages revealed that the development of such tumors may be initiated in Pax6 positive precursor cells within the ventricular zone. Finally, ex vivo cultures of murine sPNET cells were characterized by rapid tumor cell proliferation as well as sustained elevation of Wnt and Shh target genes and therefore perfectly reflected the in vivo growth properties of the tumor. CONCLUSIONS: We report on the first genetic mouse model for human sPNETs, which represents a promising tool for the development of targeted therapies against such tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0007. RESULTS OF HIGH-DOSE CHEMOTHERAPY WITH AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION IN THE TREATMENT OF PEDIATRIC BRAIN TUMORS

Asmik Gevorgian 1, Elena Morozova 1, Ilya Kazantsev 1, Tatiana Iukhta 2, Svetlana Safonova 2, Yury Punanov 2, Olga Zheludkova 3, Boris Afanasyev 1

Abstract

INTRODUCTION: Central nervous system (CNS) tumors are the second most common pediatric malignancies with an about 30% 5-year overall survival (OS) rate in relapsed group. The aim of this study was to assess the effectiveness of single or tandem high-dose chemotherapy (HDCT) with autologous hematopoietic stem-cell transplantation (auto-HSCT) in this patient group. METHOD: For 6 years, 31 patients with high-risk or relapsed medulloblastoma (N = 16), supratentorial PNET (N = 5), germinoma (N = 4), choriocarcinoma (N = 1), pineoblastoma (N = 2) and atypical teratoid rhabdoid tumor (N = 3) received HDCT with auto-HSCT after induction chemotherapy, radiotherapy, surgical treatment. At the moment of HDCT 13 patients were in complete remission (CR), 15 patients were in partial remission (PR) and 3 patients had stable disease (SD). 18 Patients received single auto-HSCT, 4 patients received tandem auto-HSCT and 9 patients received only the first of tandem. The conditioning regimens included intraventricular/intrathecal metotrexat. The mean transplanted CD34+ cell dose was 5.27 x 106/kg (1.0–8.9 x 106/kg). RESULTS: The median follow-up is 12 months (1–82). All patients with SD at the moment of auto-HSCT died of disease progression. Eight of 28 patients with CR or PR relapsed after HDCT, the other 20 patients are currently in CR. The following therapy toxicity included liver (N = 21), skin (N = 6), severe mucositis (N = 15), nausea/vomiting (N = 10), infectious (N = 18). OS in all groups was 44% and DFS was 35%. OS was significantly better among high-risk patients in 1st CR compared to patients in 2nd or following CR: 69% and 30%, respectively (p = 0,05). The same correlation was observed in DFS: 46% and 26%, accordingly (p = 0,04). CONCLUSIONS: HDCT with auto-HSCT in pediatric patients with high-risk CNS tumors may be a feasible option for patients in CR or PR after induction chemotherapy. It is ineffective as a salvage therapy in refractory patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0008. THE WIP1 ONCOGENE DRIVES METASTASIS IN GROUP 3 MEDULLOBLASTOMA

Meghan Buss 1, Marc Remke 4, Khanjan Gandhi 1, Marcel Kool 3, Paul Northcott 3, Stefan Pfister 3, Michael Taylor 4, Robert Castellino 2

Abstract

INTRODUCTION: Recent studies have suggested that medulloblastoma, the most common malignant brain tumor of childhood, comprises actually four disease variants. We have previously demonstrated overexpression of the WIP1 oncogene in Group 3 tumors, the medulloblastoma subgroup with the most aggressive clinical behavior. We have recently discovered an important interaction between WIP1 and signaling through the G protein-coupled receptor CXCR4 that promotes invasion and metastasis, which may explain the aggressive clinical behavior of WIP1 high-expressing medulloblastomas. METHOD: We examined the correlation of WIP1 expression to clinical characteristics, including survival, in a cohort of 62 patients diagnosed with medulloblastoma. We then examined gene and protein expression, as well as the interaction between WIP1 and CXCR4 signaling in Myc high-expressing, Group 3-like medulloblastoma cells containing wild-type or mutant WIP1 in vitro and in orthotopic, xenografted mouse models. RESULTS: We noted increased WIP1 in metastatic medulloblastomas, along with poor progression-free and overall survival. Microarrays identified up-regulation of metastasis genes, including CXCR4, in WIP high-expressing medulloblastomas. The CXCR4 ligand, SDF1a, activated PI3-kinase signaling and promoted growth and invasion in vitro and in an orthotopic, xenografted medulloblastoma model. Knock-down of WIP1 or CXCR4 inhibited SDF1a effects and improved survival of xenografted mice. WIP1 knock-down inhibited CXCR4 surface localization by suppressing GRK5. Restoration of GRK5 promoted Ser339 CXCR4 phosphorylation and inhibited growth of WIP1-stable medulloblastomas. GRK5 knock-down inhibited CXCR4 phosphorylation, increased CXCR4 surface localization, and promoted growth of WIP1 low-expressing medulloblastomas. CONCLUSIONS: Our results demonstrate an important cross-talk among WIP1, CXCR4, and GRK5, which may be a key determinant of the aggressive and metastatic phenotype of Group 3 medulloblastoma in children.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0009. CONGENITAL POSTERIOR FOSSA POLAR SPONGIOBLASTOMA

Jared Thompson 1, Linda Margraf 1, David Donahue 1, Hayden Head 1, Jeffrey Murray 1, Peter Burger 2

Abstract

INTRODUCTION: ‘Polar spongioblastoma' is a historical microscopic description of a tumor pattern that can be observed in a variety of high-grade pediatric and adult CNS neoplasms. It is characterized by bipolar neoplastic cells arranged in distinctive palisades. It is not recognized as a diagnostic entity in the current W.H.O. CNS tumor classification, though some argue it is indeed a unique tumor entity. METHOD: Following IRB approval, the medical record of an infant classified as having congenital posterior fossa ‘polar spongioblastoma’ was abstracted for demographic information, diagnostic details, tumor histopathology and overall clinical course. The medical literature pertaining to congenital CNS tumors, young-age medulloblastoma and ‘polar spongioblastoma’ was surveyed. RESULTS: A 3 month old presented with progressive irritability, back arching and poor feeding since birth. Scans revealed a large rim-enhancing posterior fossa mass. Gross total resection was performed. There were no metastases. The tumor was comprised nearly exclusively of palisading columns of bipolar cells with a brisk MIB-1 index (10%). There were no morphologic features of medulloblastoma or glioma. Atypical teratoid/rhabdoid tumor was excluded by strong INI-1 immunostaining. There was patchy weak synaptophysin (neuronal) and focal GFAP (glial) staining. A diagnosis of malignant ‘polar spongioblastoma’ was assigned based on the distinctive histology of this embryonic glioneuronal neoplasm. CONCLUSIONS: Whether ‘polar spongioblastoma’ is a unique tumor entity, rather than a description of tumor cell palisading that can be commonly seen in a variety of high-grade CNS tumors, is controversial. We suggest that the unique feature of virtual complete cellular palisading, in the absence of diagnostic features which would otherwise categorize the tumor into a known neuroepithelial tumor class (e.g., medulloblastoma), should prompt ongoing discussions about the nomenclature for this rare condition. The infant discussed here represents the youngest reported case of polar spongioblastoma. We suggest ongoing reporting of this condition.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0011. ABERRANT Otx2 EXPRESSION ENHANCES MIGRATION AND INDUCES ECTOPIC PROLIFERATION OF HINDBRAIN NEURONAL PROGENITOR CELLS

Matthew Wortham 1, Zach Reitman 1, Yiping He 1, Darell Bigner 1, Hai Yan 1

Abstract

INTRODUCTION: Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. METHOD: Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. RESULTS: Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. CONCLUSIONS: These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0012. PERSONALIZING THE TREATMENT FOR MEDULLOBLASTOMA: POLO-LIKE KINASE 1 (PLK1) AS A MOLECULAR TARGET FOR THE SONIC HEDGEHOG (SHH) SUBTYPE

Cathy Lee 1, Joanna Triscott 1, Colleen Foster 2, Branavan Manoranjan 3, Mary Rose Pambid 1, Abbas Fotovati 1, Rachel Berns 1, Chitra Venugopal 3, Katherine O'Halloran 1, Aru Narendran 4, Paul Northcott 5, Michael D Taylor 6, Sheila K Singh 3, Ash Singhal 2, Rod Rassekh 1, Christopher A Maxwell 1, Christopher Dunham 2, Sandra E Dunn 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common malignant brain tumor in children. There are four different molecular subtypes of MB (WNT, SHH, Group 3, and Group 4) that are used to stratify risk related to patient outcome and guide the development of targeted therapies. We recently published that Polo-Like Kinase 1 (PLK1) is a promising molecular target for glioblastoma and therefore questioned whether it too could be beneficial for MB. PLK1 is an oncogenic kinase that controls mitosis, cell cycle and proliferation making it a strong candidate for treatment of MB. METHOD: Using NanoString nCounter analysis, we subtyped 74 primary pediatric MB and measured levels of PLK1 expression. As well, a library of small molecule inhibitors were tested for efficacy again SHH MB. Both mRNA and protein levels of PLK1 were analyzed for prognostic significance. In addition, levels of PLK1 were also compared relative to normal cerebellum, neurons and astrocytes. BI2536, a PLK1 inhibitor, was assessed in self-renewal, proliferation, cell cycle arrest, and apoptosis assays. Finally, a mouse xenograft model was used to examine the efficacy of BI2536 and comparisons were made to the standard-of care chemotherapy protocol Headstart. RESULTS: Patients belonging to the SHH subtype had the poorest prognosis. PLK1 inhibitors represented 6/16 most potent inhibitors tested in the SHH MB screen. As well, both PLK1 mRNA and protein were prognostic for relapse and overall survival. PLK1 was the highest in SHH tumors and overexpressed in MB compared to normal cerebellum. We show that BI2536 has efficacy against self-renewal and proliferation in PLK1-high but not PLK1-low MB cells. In contrast, human neural stem cell growth was unaffected by BI2536. Lastly, as a single agent BI2536 extended survival in MB-bearing mice and it was comparable to the Headstart chemotherapy cocktail. CONCLUSIONS: In conclusion, patients that have tumors expressing very high levels of PLK1 are considered to be at elevated risk for relapse and death. We anticipate that PLK1 inhibitors may have fewer detrimental side-effects as it is not expressed at high levels in normal brain tissue. Therefore, it could be a great improvement to many of the chemotherapies currently being used that can often cause long-term adverse effects. These pre-clinical studies pave the way for improving the treatment of SHH MB through PLK1 inhibition.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0013. OVERCOMING RESISTANCE TO SONIC HEDGEHOG INHIBITION BY TARGETING p90 RIBOSOMAL S6 KINASE FOR THE MANAGEMENT OF MEDULLOBLASTOMA

Mary Rose Pambid 1, Rachel Berns 1, Kaiji Hu 1, Hans Adomat 2, Mani Moniri 2, Mei Yieng Chin 2, Mohammed Hessein 2, Natalia Zisman 3, Norbert Maurer 3, Christopher Dunham 4, Emma Guns 2, Sandra Dunn 1

Abstract

INTRODUCTION: Advances in molecular subtyping have allowed for a more definitive roadmap in personalizing treatment of the most common malignant pediatric brain tumour: medulloblastoma (MB). There are four molecular subtypes of MB: WNT, SHH, Group 3 and Group 4. METHOD: Based on our analysis of ∼80 pediatric MB at the British Columbia Children's Hospital, patients with the SHH have the worst prognosis when compared to the other subtypes. Despite available therapies for SHH MB, it remains most prevalent among infants with a lethal 40% recurrence rate. Screening a library of 129 compounds against cells that are resistant to SHH inhibitors, we identified BI-D1870, which inhibits RSK (p90 ribosomal S6 kinase). RESULTS: We demonstrate that BI-D1870 is effective against brain tumour-initiating cells as well as a primary human sample with no toxicity towards normal human neural stem cells at low concentrations. It induces apoptosis, restricts colony formation, sensitizes cells to chemotherapy, and crosses the blood-brain barrier as assessed by pharmacokinetic studies. CONCLUSIONS: Thus, BI-D1870 is a promising novel therapeutic agent to eliminate SHH MB. We plan to move forward with BI-D1870 into an orthotopic brain tumour model to establish its efficacy in vivo. Ultimately, our efforts are towards the translation of BI-D1870 from bench to bedside and ending the suffering caused by SHH MB.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0014. NEWCASTLE DISEASE VIRUS INDUCES IMMUNOGENIC CELL DEATH IN THE GL261 ORTHOTOPIC MOUSE MODEL

Carolien Koks 1, Steven De Vleeschouwer 1, Norbert Graf 2, Stefaan Van Gool 1

Abstract

INTRODUCTION: Glioblastoma multiforme (GBM) is classified as a WHO grade IV neoplasm and is the most frequent primary brain tumor in adults. In spite of current multimodal treatment approaches the prognosis of GBM patients remains poor, with a median overall survival of 14.6 months and a mortality rate of 88% within 3 years. Much research if performed on innovative strategies to control disease. We here explore the efficacy of oncolytic viruses (OVs) in the GL261 orthotopic mouse model. METHOD: Bl6 mice were challenged orthotopically with GL261 tumor cells. Newcastle Disease Virus (NDV) were injected once into the tumor after tumor challenge ranging from day 3 to day 7. Animals were followed for symptoms with clinical scores, and for survival. Surviving animals were rechallenged after day 60, in order to assess the induction of an immunological memory against tumor cells. RESULTS: Local injection of NDV after tumor challenge was feasible and none of the tumor challenged animals died from he technique. 45% of mice survived tumor challenge when treated with NDV versus none in control with tumor challenge alone. NDV injected at time of minimal disease (day 3) or at time of bulky tumor (day 7) induced similar survival data. Subsequent tumor challenge in surviving mice resulted in 80% survival, suggesting that most of the mice were immunized after NDV therapy. NDV therapy failed to prolong survival of immunodeficient glioma-bearing mice. CONCLUSIONS: For the first time, we describe that NDV has an activity against GL261 tumor cells, evidenced in the orthotopic mouse model. NDV therapy not only reduces tumor burden but induces also long-term survival. For the latter, the induction of an anti-glioma immune response is necessary. The model will allow us to study potential synergy between NDV therapy and dendritic cell therapy.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0015. DEREGULATION OF TISSUE FACTOR BY A PUTATIVE ONCOGENIC microRNA, miR-520g, IN EMBRYONAL BRAIN TUMOUR CELLS

Esterina D'Asti 1, Annie Huang 2, Andrey Korshunov 3, Stephen Pfister 3, Janusz Rak 1

Abstract

INTRODUCTION: Embryonal tumours with multilayered rosettes (ETMR) demonstrate a unique amplification of the C19q13.42 genomic locus, leading to overexpression of the related microRNA (miR) cluster, including the functionally important miR-520g. ETMRs exhibit significant vascular pathology including intravascular thrombosis and angiogenesis, which are often a consequence of oncogenic deregulation of tissue factor (TF). TF acts as an essential receptor for coagulation factor VIIa and thereby mediates both clotting and pro-angiogenic intracellular signaling. We hypothesized that in ETMR the amplified miR-520g may be a part of the oncogenic circuitry that controls TF resulting in perturbations in vascular homeostasis and increased disease aggressiveness. METHOD: TF immunopositivity was evaluated for ETMR tumours with C19q13.42 amplification. To mimic this effect medulloblastoma (MB) and glioblastoma (GBM) cell lines were transfected to overexpress miR-520g (DAOY, UW228, U373vIII). The cells were also treated with 5-AZA-2'deoxycytidine to unmask endogenous miR-520g. TF mRNA and protein levels were assessed in the cellular and extracellular vesicle (EV) fraction under different conditions. Bioinformatics, LNA miR inhibitors, and luciferase reporter plasmids were used to assess the regulation of TF by miR-520g. Biological implications were studied by assessing TF procoagulant activity and levels of TF target genes (plasminogen activator inhibitor-1, PAI-1). RESULTS: TF is expressed in pediatric embryonal brain tumours, but expression is low in C19q13.42-amplified ETMR. Moreover, mir-520g is methylated in MB and GBM cells expressing high levels of TF. Enforced expression of miR-520g reduces cellular and EV-associated TF levels in vitro and in DAOY tumour xenografts. Luciferase assays indicate that this effect is mediated by miR-520g binding sites in the TF-3'UTR sequence, and is reversible by the respective anti-mir. Growth factors (TGFα) attenuate miR-520g effects. MiR-520g lowers the TF-dependent procoagulant activity of MB cells and their VIIa-induced production of PAI-1. CONCLUSIONS: In spite of the absence of clinically manifest thrombosis, pediatric brain tumours are able to activate the coagulation pathway by overexpression of TF. Ours is the first study that examines this as a function of miRs and shows TF downregulation by miR-520g, a putative oncomir in ETMR. The significance of this change for ETMR pathogenesis is presently unclear. Since miR-520g is methylated in other types of brain tumour cells (MB, GBM) where TF levels are normally high, we suggest that this silencing may be indicative of a tumour suppressive activity. We propose that oncomirs may control brain tumour coagulome.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0016. LATE RECURRENCE OF SUPRATENTORIAL EPENDYMOMA WITH HISTOLOGIC PROGRESSION: CASE ILLUSTRATION

William Gump 1, Thomas Moriarty 1

Abstract

INTRODUCTION: Supratentorial ependymoma is a rare malignancy with poor long-term prognosis due to strong tendency for local and remote recurrence. Outcomes are best when gross total resection is followed by radiation. We report the case of an explosive tumor recurrence following an 8 year disease free interval. METHOD: A 3 year old girl presented with seizures and a frontoparietal brain tumor. She underwent gross total resection of a WHO grade II ependymoma, then conformal radiation. Surveillance MR imaging up to 7 years 7 months from the time of resection showed no disease recurrence. 8 years 2 months after resection, she presented with headaches, left hemiparesis, and large local tumor recurrence. On reoperation, histology was WHO grade III anaplastic ependymoma. Four months later, biopsy of a small area of new enhancement at the margin of the resection cavity revealed no malignancy. She has been disease free for two years. RESULTS: Outcomes studies in patients with ependymoma show very similar overall survival and progression-free survival at 5 and 10 years following treatment. Median time for progression or relapse has been found to be about 2 years. Prognosis is similar between WHO grade II and grade III histologies, worse with younger age at initial presentation, and worse with shorter time to relapse. Disease progression or recurrence first seen at 5 to 10 years after initial treatment is very rare. Factors which could lead to delayed recurrence after gross total resection are unknown. CONCLUSIONS: Tumor surveillance following optimal treatment for supratentorial ependymoma should be continued for at least 10 years. Further study is needed to identify patients who may be at risk for delayed recurrence despite gross total resection.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0017. CONTROL OF REFRACTORY EPILEPSY AFTER SUBTOTAL RESECTION OF LOW GRADE GLIOMA

William Gump 1, Karen Skjei 1, Shefali Karkare 2

Abstract

INTRODUCTION: Brain tumors have a well-established association with epilepsy, with low grade gliomas known to be particularly epileptogenic. Reports on seizure outcomes from surgery on lesional epilepsy consistently cite extent of resection as a significant predictor of success. Unfortunately, not all tumors are resectable. We report the case of a patient whose pharmacoresistant seizures were ultimately controlled after tailored resection of a large diffuse protoplasmic astrocytoma. METHOD: A 4 year old girl presented with three months of generalized seizures. Neurologic exam was nonfocal. MRI of the brain revealed an extensive diffuse nonenhancing area of abnormal signal involving the right frontal and temporal cortex and subcortical structures. There was minimal mass effect and no restricted diffusion. Initial diagnosis at biopsy was nonspecific low-grade glioma. Medical control of the patient's seizures gradually deteriorated despite polypharmacy and no radiographic progression. She returned to surgery eight months later for electrocorticography-guided temporal lobectomy and extratemporal resection, yielding the diagnosis of protoplasmic astrocytoma. Seizure frequency decreased to zero over the following three months. RESULTS: Low grade gliomas are the most common neoplastic pathology associated with pharmacoresistant epilepsy. Better seizure outcomes have been consistently found in patients undergoing gross total resection. Electrocorticography has not been shown to improve outcomes, but has not been used consistently in subtotal resections. In patients with unresectable lesions, factors have not been identified which can improve the potential for good seizure outcome with surgical intervention. CONCLUSIONS: Pharmacoresistant epilepsy is common in patients harboring brain tumors. For lesions in which gross total resection is not possible, further study is needed to ascertain strategies which maximize the chances for a seizure-free outcome.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0018. METHYLATION OF A SPECIFIC REGION IN THE hTERT PROMOTER IS A CANCER SIGNATURE WHICH CORRELATES WITH TUMOR GRADE AND PATIENT SURVIVAL

Pedro Castelo-Branco 1, Sanaa Choufani 2, Stephen Mack 1, Denis Gallagher 3, Cindy Zhang 1, Diana Merino 2, Jonathan Wasserman 2, Marcel Kool 5, David TW Jones 5, Sidney Croul 1, Cynthia Hawkins 1, Johann Hitzler 4, Jean CY Wang 6, Sylvain Baruchel 4, Peter B Dirks 1, David Malkin 2, Stefan Pfister 5, Michael D Taylor 1, Rosanna Weksberg 2, Uri Tabori 1

Abstract

INTRODUCTION: Defining grade of malignancy and disease progression is a major goal in oncology. Since telomere maintenance is a hallmark of cancer, we examined whether promoter methylation of hTERT, the catalytic subunit of telomerase, can be a biomarker for cancer malignancy, response to therapy and patient outcome. METHOD: Whole genome methylation arrays (discovery cohort n = 280). Sequenom and pyrosequencing of the hTERT promoter (validation cohort, n = 319) were performed on tumor samples and normal tissues. Correlation between hTERT promoter methylation, telomere maintenance and patient outcome was performed. RESULTS: We identified a specific region upstream-of-the transcription-start-site (UTSS) of hTERT that is hypermethylated in malignant tumors and is unmethylated in normal tissues and in low grade tumors. Malignant-gliomas without UTSS hypermethylation lacked hTERT expression and revealed an alternative lengthening of telomeres phenotype. UTSS methylation was able to identify which low-grade tumors would progress to high-grade ones. Ependymomas with and without UTSS methylation had 5-year overall survival of 51 + /-10% and 95 + /-5% respectively (p = 0.0008). UTSS methylation predicted which neural tumors would respond to targeted therapy with telomerase inhibitor. Combination of telomerase inhibition and demethylation resulted in synergistic tumor growth-arrest in glioma cells. CONCLUSIONS: Hypermethylation of a specific region in the hTERT promoter positively correlates with higher hTERT expression, tumor progression and poor prognosis. This signature may also represent a diagnostic tool and a therapeutic target for multiple cancers.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0019. EXPLORING THE FUNCTIONAL ROLES AND THERAPEUTIC POTENTIAL OF TARGETING POTASSIUM CHANNEL EAG2 IN MEDULLOBLASTOMA GROWTH AND METASTASIS

Xi Huang 1, Ye He 1, Adrian M Dubuc 2, Rintaro Hashizume 3, Wei Zhang 1, Samantha Stehbens 4, Susan Younger 1, Suzanne Barshow 1, Sijun Zhu 1, Xiaochong Wu 2, Michael D Taylor 2, William A Weiss 3, C David James 3, Marc A Shuman 5, Yuh Nung Jan 1, Lily Y Jan 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric CNS tumor, and is characterized by rapid progression and tendency to spread along the leptomeninges of the brain and spinal cord. Standard-of-care treatment with surgery, radiation and chemotherapy typically results in serious cognitive and neuroendocrine deficits that substantially impact quality of life. It is therefore critically important to identify novel targets that drive MB cell growth and tumor progression. The contribution of ion channels towards MB tumorigenesis and progression is essentially unexplored. Here we have investigated the functional roles and therapeutic potential of targeting potassium channel Eag2 in MB growth and metastasis. METHOD: We conducted a genome-wide gene expression microarray survey of mouse Shh-MBs and found that the expression of voltage-gated potassium channel Eag2 was consistently elevated. Using qPCR, IHC and bioinformatic approaches we confirmed EAG2 overexpression in a subset of human MBs across molecular and histological subgroups. We studied the functional roles of EAG2 using electrophysiology, molecular biology, cell biology, and xenograft approaches following specific RNAi knockdown of Eag2 expression. We also crossed Eag2 knockout mice with transgenic mice that develop spontaneous MBs and determined changes in tumor progression. Finally, we studied the evolutionarily conserved role of eag channel in Drosophila melanogaster. RESULTS: We determined that EAG2 expression is elevated across different molecular and histological subtypes of MB. EAG2 knockdown significantly impaired tumor growth in vitro and in vivo. Mechanistically, we show that EAG2 controls mitotic entry and tumor growth by regulating cell volume dynamics. Intriguingly, we found that EAG2 also promotes MB cell migration and tumor metastatic potential. In addition, we determined that EAG2 deficiency or pharmacological inhibition reduces MB cell proliferation and migration via effect on the p38 MAPK pathway. Importantly, we found that the brain tumor-specific function of eag potassium channel is evolutionarily conserved across distant organisms. CONCLUSIONS: Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which acts through the p38 MAPK pathway, and establishes clinical relevance for targeting this ion channel in human MBs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0020. DELAYED FUNCTIONAL PATENCY OF ENDOSCOPIC THIRD VENTRICULOSTOMY WITH RADIATION-INDUCED ALLEVIATION OF MASS EFFECT: CASE ILLUSTRATION

William Gump 1, Ian Mutchnick 1

Abstract

INTRODUCTION: Endoscopic third ventriculostomy (E3V) is an established option for treatment of obstructive hydrocephalus and is often performed concurrentl with biopsy of an obstructing mass lesion. Delayed failure is a known complication. We report the case of a patient whose E3V became functionally patent in a delayed fashion, three weeks postoperatively, when mass effect on the brainstem had been adequately relieved. METHOD: A 13 year old boy presented to the emergency department with symptomatic obstructive hydrocephalus from a pineal region tumor. He underwent endoscopic biopsy, E3V, and external ventricular drain (EVD) placement. Pathology was consistent with pure germinoma. The patient returned to surgery four days later for a secondary E3V. He remained drain-dependent, and initiated radiation therapy on postoperative day #7. Placement of a ventriculoperitoneal shunt was deferred while the patient was still receiving radiation. RESULTS: EVD output declined from 300 cc/day to 100 cc/day by the 11th day of radiation therapy. On postoperative day #22 the EVD was clamped, and the patient remained asymptomatic. A new brain MRI the following day showed significantly decreased size of the germinoma along with flow artifact across the floor of the third ventricle, and his EVD was removed. There has been no tumor progression and the patient's hydrocephalus has not recurred over the following two years. CONCLUSIONS: Resolving mass effect from a rapidly shrinking pineal region tumor can delay relief of hydrocephalus from E3V, but has the potential to ultimately result in shunt independence.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0021. GENOMIC GAIN AND OVEREXPRESSION OF KIF14 IN MEDULLOBLASTOMA

Kay Ka-Wai Li 1, Jesse Chung-Sean Pang 1, Ho-Keung Ng 1

Abstract

INTRODUCTION: Despite recent advances in multimodality therapy, one-third of medulloblastoma (MB) patients remain incurable. Thus, novel therapies targeting the underlying molecular pathogenesis of MB are needed. Kinesin family member 14 (KIF14) is a mitotic kinesin that has been implicated in tumorigenesis. Upregulation of KIF14 is associated with poor clinical outcomes in multiple cancers. The objectives of this study were to determine the expression of KIF14 in MB and to delineate the functional roles KIF14 in MB pathogenesis. METHOD: The transcript levels of KIF14 were examined by quantitative real-time RT-PCR. Genomic copy number status of chromosome 1q was determined by fluorescent in situ hybridization (FISH). Two gene-specific siRNAs were employed to deplete KIF14 expression in 2 MB cell lines, DAOY and ONS76. Cell cycle profile, cellular proliferation, and DNA fragmentation was employed to determine the effects of silencing KIF14 in MB cells. In addition, MB cells transfected with siRNAs against KIF14 were injected into the cerebella of athymic nude mice to determine the incidence of tumor formation. RESULTS: Overexpression of KIF14 was identified in 22/28 of MB and 7/7 cell lines. Eight of the tumors displayed gain of chromosome 1q. Tumors with 1q gain had significantly increased KIF14 expression compared with those without (p = 0.0359). Silencing KIF14 led to inhibition of cell proliferation by 53-80%. KIF14-depleted cells showed an accumulation of cells in sub-G1 phase and an increase in DNA fragmentation by 20-33 -fold. When KIF14-depleted cells were injected into cerebella of the mice, no tumor was produced. In contrast, tumors were formed at a frequency of 5/12 (41.7%) when mice were injected with negative control-transfected MB cells. CONCLUSIONS: Overexpression of KIF14 is a frequent event in MB. Silencing KIF14 reduced cell proliferation by induction of apoptosis in vitro, and suppressed tumor formation in vivo. Together, these data suggest targeting KIF14 may be a novel potential therapeutic target of MB.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0022. CLINICO-RADIOLOGIC CHARACTERISTICS AND NEUROCOGNITIVE ASSESSMENT OF LONG-TERM SURVIVORS OF DIFFUSE INTRINSIC PONTINE GLIOMA

Sadhana Jackson 1, Zoltan Patay 1, Robyn Howarth 1, Atmaram Pai 1, Arzu Onar-Thomas 1, Amar Gajjar 1, Alberto Broniscer 1

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is the deadliest central nervous system tumor in children despite treatment with radiation therapy (RT) with or without chemotherapy. METHOD: We reviewed the medical records of all surviving patients with DIPG treated at our institution between October 1, 1992 and May 31, 2011. All surviving patients underwent detailed neurocognitive assessment adapted according to their age and functional level. RESULTS: Five of 191 patients treated were surviving at a median of 9.3 years from diagnosis. Four patients had clinical and radiologic characteristics at diagnosis associated with improved outcome. Chemotherapy varied among these patients yet all received RT. Neurocognitive assessments were obtained after a median interval of 7.1 years. One patient diagnosed at age 16 had normal range scores, yet all others displayed global intellectual deficits. Two patients aged 7.3 and 8.4 years at diagnosis had cognitive function in the borderline and mild retardation range, respectively, despite almost complete neurologic recovery. Two patients experienced disease progression at 8.8 and 13 years after diagnosis. CONCLUSIONS: A minority of children with DIPG experienced long-term survival with currently available therapies. These patients remained at high risk for tumor progression even after long follow-ups. The observation of major cognitive deficits even among children diagnosed at older ages despite a normal neurologic function was concerning.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0023. INTRACRANIAL XENOGRAFTING OF EXTRANEURAL DISSEMINATED ANAPLASTIC MEDULLOBLASTOMA IN IMMUNOCOMPROMISED MICE AND SUBSEQUENT IN VIVO BIOLUMINESCENCE IMAGING

Sebastian Maximilian Dietl 1, Matthias Wölfl 1, Andreas Beilhack 2, Simone Riedel 2, Camelia Monoranu 3, Susanne Dietl 1, Giles Hamilton Vince 4, Matthias Eyrich 1, Paul-Gerhardt Schlegel 1

Abstract

INTRODUCTION: Medulloblastoma (MB) represents the most common malignant brain tumor in children. Although survival has significantly improved within the last decades, prognosis for high risk patients still remains poor. Criteria associated with poor clinical outcome are: age ≤3 years, metastasis, subtotal resection, anaplastic histology and c-myc amplification. We isolated MB cells from the pleura effusion of a 2 year old boy suffering from a c-myc positive anaplastic MB that also showed dissemination via the cerebrospinal fluid. To further investigate the biology of these "high risk" cells, we established a xenogenic mouse model with the ability for bioluminescence imaging (BLI). METHOD: MB cells, transduced with a lentiviral vector coding for enhanced green fluorescent protein (eGFP) and firefly luciferase (FLuc), were evaluated by fluorescence microscopy, flow cytometry and in vitro BLI. For intracranial grafting transduced cells were injected i) infratentorially and ii) supratentorially into the brains of immunocompromised mice. Tumor growth was evaluated by clinical surveillance and kinetic in vivo BLI. Tumors were histologically analyzed. RESULTS: After lentiviral transduction, expression of eGFP could be easily detected by fluorescence microscopy and flow cytometry. Analyzing FLuc activity by limited dilution assay and BLI revealed emission of 483 photons/second/cell. When implanting the cells into the brains of immunocompromised mice, there was a 100 % penetrance. As few as 500 cells were capable to form tumors. BLI showed a continuously increasing signal after supra- as well as infratentorial grafting. Histology and immunohistochemistry revealed that tumors were identical to the primary tumor. They were anaplastic MBs and showed a high tendency to invade and spread through the cerebrospinal fluid. CONCLUSIONS: We established a highly reproducible anaplastic MB model that closely resembles the morphological (i.e. anaplasia) and biological (i.e. dissemination) features of the primary tumor. Because of lentiviral transduction, noninvasive imaging of in vivo cell growth is possible thus providing an ideal tool for monitoring tumor biology or treatment effects.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0025. RADIOPROTECTION OF BRAIN WHITE MATTER BY THE CATALYTIC MnSOD MIMIC / ANTIOXIDANT, BMX-001

Douglas Weitzel 1, Kathleen Ashcraft 1, Chunlei Liu 1, Wei Li 1, Anne Buckley 1, Ramona Rodriguez 1, William Wetzel 1, Ivan Spazojevic 1, Artak Tovmasyan 1, Katherine Peters 1, Ines Batinic-Haberle 1, Mark Dewhirst 1

Abstract

INTRODUCTION: Cranial irradiation is a proven therapy for primary and metastatic brain tumors that increases cancer patient survival. A major drawback of radiotherapy (RT), however, is long-term cognitive loss that affects the quality of life of these survivors. Radiation-induced oxidative stress in normal brain tissue is thought to contribute to neurocognitive decline. METHOD: We evaluated the effectiveness of a novel catalytic mimic of Mn superoxide dismutase (MnSOD), BMX-001 (MnTnBuOE-2-PyP+5), to provide neuroprotection following 8 Gray of cranial irradiation. C57BL/6J mice were pretreated with BMX-001 by twice-daily injections of 1.5 mg/kg for one week prior to and one month following irradiation. The BMX-001 dose was then reduced to 0.5 mg/kg/day for another month and then discontinued for a third month post-irradiation. Mouse brains were evaluated by MRI (radial diffusion, susceptibility and functional anisotropy) using a 9T Oxford vertical bore magnet at 3 months post RT. RESULTS: Concentrations of drug in the brain averaged 25nM, which is within the therapeutic dose range, based on other studies in stroke and spinal cord injury. Mice treated with RT + BMX-001 exhibited strong MRI evidence for myelin preservation in the corpus callosum and anterior commissures compared with controls that received saline + RT. The saline + RT groups exhibited neurocognitive deficits using the rotorod, an indicator of motor learning. These deficits were ameliorated in groups treated with RT + BMX-001. Wheel running tests revealed a significant decrease in running distance for saline + RT, compared with BMX-001 + RT. Interestingly, circadian rhythms of saline + RT groups were shorter than all other groups. CONCLUSIONS: These data demonstrate that BMX-001 is neuroprotective as assessed by MRI evaluation of white matter integrity and neurocognition. BMX-001 treatment showed no overt toxicities despite the extended treatment schedule. Therefore BMX-001 is potentially attractive candidate for prevention of neurocognitive losses post RT in future studies in pediatric patients with CNS tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0026. BLOCKING THE ANTI PHAGOCYTIC SIGNAL PROVIDES A COMMON AND EFFICACIOUS TREATMENT MODALITY FOR PEDIATRIC BRAIN TUMORS

Sharareh Gholamin 2, Siddhartha Mitra 1, Chase Richard 2, Doo-Sik Kong 2, Achal Achrol 2, Jun Jae Shae 2, Michelle Monje-Deisseroth 4, Yoon-Jae Cho 3, Irving Weissman 1, Samuel Cheshier 2

Abstract

INTRODUCTION: Extensive molecular characterization of Brain tumors has proven valuable for classification, risk stratification and outcome prediction for current treatment. However the standard of care has not improved prognosis and carries an increased risk of cognitive impairments for children. A characteristic feature of tumor progression and recurrence is its ability to evade the immune system. We hypothesized that by disrupting the interaction between the cell surface antigen CD47 and its binding partner Sirpa we can induce the innate immune system to attack and remove the tumor by enhancing the ability of macrophages to phagocytose brain tumor cells. METHOD: We looked at CD47 expression in freshly isolated patient and postmortem samples from 5 different tumor types; Diffused Intrinsic Pontine Glioma, Medulloblastoma, Ependymoma, PNETs and ATRTs. We next tested the hypothesis that blocking CD47-Sirpa interaction induced phagocytosis in an in-vitro phagocytosis assay. Finally we established orthotopic xenografts models from primary patients samples in immune compromised mice and treated them with anti-CD47 humanized antibody, which is currently being developed for clinical trials in hematopoietic and non-CNS malignancies. RESULTS: CD47 expression was upregulated in all tumor types and was present in >90% of the cells in high grade tumor. Increased CD47 expression was observed in CD15+ and CD133+ putative cancer stem cell population. Blocking the CD47-Sirpa interaction increases tumor phagocytosis by macrophages in-vitro. Systemic treatment with anti-CD47 antibody significantly reduced tumor burden in an orthotopic xenograft setting. CONCLUSIONS: Anti-CD47 therapy is a viable and effective treatment modality for pediatric high grade brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0027. GENETIC AND HISTOPATHOLOGICAL SPECTRUM OF PAEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMAS

Pawel Buczkowicz 1, Patricia Rakopoulos 1, Eric Bouffet 1, Andrew Morrison 1, Ute Bartels 1, Oren Becher 2, Cynthia Hawkins 1

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a devastating malignancy with poor prognosis, no effective therapy and a mean survival of ∼10 months. In the majority of cases diagnosis is based exclusively on MRI findings, resulting in a scarcity of pre-treatment specimens available to study. Focal radiation is currently the standard of care with many chemotherapeutic agents showing little success thus far. To try to improve our ability to treat these patients our group has developed an autopsy-based protocol to investigate the biology of DIPG and correlate this with histologic and clinical features. METHOD: Here we report the histologic spectrum of seventy-one DIPG and its relation to genetic events including mutations, structural variants and copy-number alterations. All cases were reviewed for histologic diagnosis and classified according to WHO criteria. Sixty patients had high-grade-astrocytomas (grade-III/IV), eight had grade-II-astrocytomas, and three had features of primitive neuroectodermal tumour (PNET). To identify genetic alterations underlying DIPG we performed deep sequencing of 35-tumour normal pairs (20 whole genomes and 15 whole exomes) and integrated this data with SNP copy number for 40 DIPGs. RESULTS: There was no correlation between histologic grade and survival. Approximately 1/3 of patients had leptomeningeal spread of their tumour. Diffuse invasion of the brainstem, spinal cord and thalamus was common with some cases showing spread as distant as the frontal lobes. Sixty-eight percent and patients had K27M-H3 mutations. Recurrent copy number alterations and structural variants corresponding to histone mutational status were identified including those involving PDGFRA, MYC and MYCN. An association between higher age of diagnosis and ALT (alternative lengthening of telomeres) was observed (ALT: 11.25 ± 3.27 vs non-ALT 5.91 ± 2.40; p = 0.0001). ATRX mutations were present in 1/3 of ALT positive patients. CONCLUSIONS: DIPG tumour histology is very heterogeneous with many showing focal GBM areas intermixed with areas of grade-II or grade-III histology, which has potential implications for biopsy. Histone mutations were more homogenously present and were found in low grade tumours with poor clinical outcome. The frequency of leptomeningeal disease in this patient group suggests that focal radiation may be inadequate for some of these patients. Our findings suggest that new therapeutic approaches need to incorporate both histopathological and molecular data, including histone mutational status at biopsy in order to achieve maximum benefit for DIPG patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0028. TRANSCRIPTOSOMIC PROFILING AT IGF-2 PROMOTERS THROUGH BIOTINYLATED-DNA ‘FISHING’; ANALYSIS

Abhinav Dey 1, Anna Kenney 1

Abstract

INTRODUCTION: Medulloblastomas have four molecularly distinct subclasses. Tumors of the subclass marked by over-expression and amplification of Sonic hedgehog (Shh) pathway components and targets also display high levels of insulin-like growth factor-2 (IGF2). In mouse models for Shh-medulloblastomas, IGF2 is required for tumor formation, growth, and metastases. We showed that YAP over-expression induces IGF2 expression as a part of YAP's radiation-resistance program in mouse Shh-medulloblastomas and in cerebellar granule neuron precursors (CGNPs). IGF2 and its regulatory program may represent a therapeutic target in medulloblastoma, but the mechanism of IGF2 induction downstream of YAP is not well understood. METHOD: The anomalous loss of IGF2 imprinting in the human fetal brain is intriguing and exemplifies the complexity of the IGF2 gene's regulation. Although CTCF mediates allele-specific expression at the IGF2/H19-imprinted locus in both mice and humans, subsequent evidence suggests that CTCF binding at the IGF2/H19 imprinting control region is insufficient to regulate IGF2/H19 expression in human tissues. This makes a compelling case for studying the transcriptosome at the different IGF2 promoters. We have employed biotinylated-DNA ‘fishing’ combined with proteomics to delineate the transcriptosomes at the IGF2 promoters in medulloblastoma cells, and directly validated them using medulloblastoma cell-derived material. RESULTS: This effort reveals the transcription factors and co-factors (enhancers/repressors) associated with the IGF2 transcriptosome. CONCLUSIONS: These components of the IGF2 transcriptosome may be regulating the preferential expression of IGF2 from promoter P3 and the simultaneous silencing at the remaining promoters in Shh-associated medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0029. VALIDATION OF THE HGG-IMMUNO RPA MODEL IN A NEW COHORT OF PATIENTS WITH RELAPSED MALIGNANT GLIOMA TREATED BY ADJUVANT POSTOPERATIVE DENDRITIC CELL VACCINATION

Stefaan Van Gool 1, Femke Pauwels 1, Steven De Vleeschouwer 1

Abstract

INTRODUCTION: Immunotherapy with autologous mature dendritic cells loaded with autologous tumor lysate (DCm-HGG-L) is an innovative treatment for patients with high grade glioma (HGG). We run the HGG-IMMUNO-2003 cohort comparison study to treat children and adults with relapsed HGG with immunotherapy after new (sub)total resection. We published recently the importance of the clinical risk profiles for prognostic patient counseling for participants to the immunotherapy trials (PubMedID 22565485). The data were derived from patients treated in cohorts A-D. We now aim to validate the HGG-IMMUNO RPA model in the E cohort of patients. METHOD: Patients were included after subtotal resection of the relapsed tumor, confirmed with postoperative MRI, central pathology confirmation and quick tapering off of corticosteroids. Tumors were conserved sterile, dry and frozen at -80°C for preparing the lysate with snap freeze/thaw cycles and subsequent 60 Gy irradiation. Dendritic cells (DC) were differentiated out of monocytes, obtained with elutriation of the leukapheresis product, in tissue bags in the presence of GM-CSF/IL-4 for 7 days. DCs were loaded with lysate (HGG-L) and matured with IL-1b/TNF-a for two days. They were injected intradermally in imiquimod-prepared skin at weeks 1/2/3/4/8/12/16 and then each 3 months. RESULTS: HGG-IMMUNO RPA data were available for 92 adults aged 17-75y (median 49). Patients were treated postoperatively with a median of 6 vaccines (1-23). After up to 4 weekly induction vaccines with DCm-HGG-L, boost vaccines consist of only HGG-L (weeks 8/12/6/ + 12). Median PFS and OS were 3.5 and 11 months with a 2-y OS of 17%. Patients were classified in HGG-IMMUNO RPA classes I (n = 8), II (n = 23), III (n = 45) and IV (n = 16). Median PFS and OS were respectively 3.7, 2.9, 4.1, 3.3 and 16,3, 14.7, 10.4 and 9.4 months (log-rank: p = 0.048). CONCLUSIONS: Although long-term follow up of the E cohort is still short, the HGG-IMMUNO RPA model could already significantly distinguish the median OS for patient subgroups with relapsed HGG treated in the E cohort. These data together with the published data strongly support the HGG-IMMUNO RPA model to describe patient groups treated with immunotherapy, in order to appropriately evaluate the effects of immunotherapy. The model allows a more accurate interpretation of immunotherapy treatment results amongst different research groups and opens the possibility for appropriate stratification of randomised clincial trials in future. Similar modelling is urgently needed for pediatric patients with HGG.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0030. TELOMERASE INHIBITION INDUCES GROWTH ARREST IN PAEDIATRIC EPENDYMOMA

Mark Barszczyk 1, Pawel Buczkowicz 1, Pedro Castelo-Branco 1, Stephen Mack 1, Kathleen Nethery-Brokx 1, Andrew Morrison 1, Michael Taylor 1, Peter Dirks 1, Uri Tabori 1, Cynthia Hawkins 1

Abstract

INTRODUCTION: Ependymomas represent the third most common paediatric brain tumour, yet effective therapeutics are lacking and 5-year survival rates remain poor at approximately 50%. Previous studies have shown that over 50% of paediatric ependymomas possess active telomerase, an enzyme that permits a limitless growth potential through the prevention of telomere erosion and subsequent senescence. Since telomerase is present in the majority of ependymomas and absent in the majority of somatic cells, telomerase inhibition represents an ideal therapeutic strategy for telomerase-positive paediatric ependymomas. We hypothesize that inhibiting telomerase will induce growth arrest in paediatric ependymoma cell and animal models. METHOD: Paediatric ependymoma cell lines (R254, BXD-1425EPN) and tumour initiating cells (TICs) (E520) were treated with the telomerase inhibitor Imetelstat in parallel with untreated and mismatch control until growth arrest was observed. Throughout treatment, a number of parameters were assessed including senescence (ß-galactosidase), apoptosis (TUNEL), telomerase activity (telomere repeat amplification protocol) and telomere length (telomere restriction fragment assay). To study telomerase inhibition in vivo, subcutaneous injections of 5.0x104 E520 cells were performed and mice were treated with either PBS, mismatch control or Imetelstat (3x/week) for 4 weeks (N = 6 mice/group). Tumour growth was measured with calipers and tumours were weighed upon sacrifice. RESULTS: Imetelstat treated R254 cells showed a reduced proliferative rate following 6 weeks of treatment and total growth arrest following 15 weeks of treatment. This observed growth arrest was associated with a marked inhibition of telomerase activity, shortened telomeres, an 80% increase in senescence and 20% increase in apoptosis. BXD-1425EPN cells treated with Imetelstat exhibited drastically reduced growth associated with telomerase inhibition and a 50% increase in senescence, while E520 TICs have thus far shown a reduced growth rate. In vivo, Imetelstat reduced tumour volume by 40% and tumour mass by 35% compared to PBS controls following 4 weeks of treatment. CONCLUSIONS: These findings demonstrate that telomerase inhibition can effectively reduce paediatric ependymoma growth both in vitro and in vivo. Future investigations are aimed at combining telomerase inhibition with other therapeutics to induce growth arrest earlier than with telomerase inhibition alone. Since children harboring telomerase-positive ependymomas exhibit a significantly worse prognosis than those lacking telomerase, telomerase inhibition may serve as a promising therapeutic approach for paediatric ependymoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0031. DEVELOPMENT OF A NOVEL, SINGLE-CHAIN, TRISPECIFIC IMMUNOTOXIN, D2C7-Mel-14-PE38KDEL FOR TARGETED PEDIATRIC BRAIN TUMOR THERAPY

Vidyalakshmi Chandramohan 1, Stephen T Keir 1, Xuhui Bao 1, Ira H Pastan 2, Chien-Tsun Kuan 1, Darell D Bigner 1

Abstract

INTRODUCTION: More than 4,200 new cases of pediatric brain tumors are diagnosed every year in the US. Brain tumors are genetically heterogeneous and are often difficult to treat with a single therapeutic agent. Hence, combination therapies targeting multiple tumor-associated antigens are being widely investigated. D2C7, a murine monoclonal antibody (mAb), recognizes both the EGFRwt and the tumor-specific EGFRvIII receptors, two antigens that are highly expressed in glioblastoma multiforme (GBM). The tumor antigen chondroitin sulfate proteoglycan (CSPG) is overexpressed in gliomas and is recognized by the mAb Mel-14. METHOD: The reactivity of D2C7 and Mel-14 antibodies were tested in cells isolated from pediatric GBM xenografts, D212MG, D456MG, D2159MG, D2322MG, H2360MG, by flow cytometry. A novel, recombinant, trispecific single-chain antibody tandem variable region fragment (sctaFv), D2C7-Mel-14, was cloned from the D2C7 and Mel-14 mAbs and fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (D2C7-Mel-14-PE38KDE). In vitro cytotoxicity was measured against CSPG-expressing pediatric xenograft H2224MG cells, EGFRwt-overexpressing 43 GBM xenograft cells, and an EGFRvIII-transfected NR6M cell line. RESULTS: FACS analysis revealed Mel-14 and D2C7 mAb reactivity to be 27–99% and 12–99%, respectively in the pediatric GBM xenografts. The D2C7-Mel-14-PE38KDEL was cytotoxic against CSPG-expressing H2224MG cells, with an IC50 in the range of 6.8 ng/mL, which was 12-fold lower than that of the monospecific IT, Mel-14-PE38KDEL (IC50 85 ng/mL). The IC50 of D2C7-Mel-14-PE38KDEL was 4-fold higher than that of the monospecific IT dsD2C7-PE38KDEL (IC50 4.1 vs 1.0 ng/mL) against the EGFRvIII-transfected NR6M cell line. Again, the cytotoxicity of D2C7-Mel-14-PE38KDEL was 4-fold higher than that of dsD2C7-PE38KDEL against EGFRwt-overexpressing 43 GBM xenograft cells (IC50 9.0 vs 2.2 ng/mL). CONCLUSIONS: The D2C7-Mel-14-PE38KDEL is a novel, trispecific IT exhibiting significant cytotoxicity against all three brain tumor antigens, EGFRwt, EGFRvIII and CSPG. We are in the process of testing the in vitro and in vivo efficacy of the D2C7-Mel-14-PE38KDEL against EGFRwt-, EGFRvIII-, and CSPG-expressing pediatric brain tumor xenograft models.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0032. MUTANT HISTONE H3.3 INDUCES CHANGES IN KEY EPIGENETIC MODIFICATIONS IN PEDIATRIC GLIOBLASTOMA

Sebastian Bender 1, David Jones 1, Marcel Kool 1, Dominik Sturm 1, Andrey Korshunov 2, Peter Lichter 3, Stefan M Pfister 1

Abstract

INTRODUCTION: Chromatin structure is tightly regulated by a diverse set of mechanisms including covalent modification of histone tails and incorporation of non-canonical histone variants. Deregulated epigenetic modifications and altered chromatin configuration have been found to drive cells into cancerous development. Besides numerous mutations targeting the enzymatic machinery of histone modifications in multiple cancer types, it has been shown that pediatric glioblastomas (pedGBMs) harbor mutations within the histone variant H3.3. These recurrent somatic mutations lead to the substitution of amino acids at lysine 27 (K27M) or glycine 34 (G34R/V) of histone H3.3 - at or near sites of key epigenetic modifications. METHOD: In order to identify the complex pattern of alterations to posttranslational histone modifications (PTMs) in primary H3.3-mutated pedGBM samples, we performed immunohistochemistry on our tissue microarray containing 128 tumor cores. In addition, histone extracts of mutant H3.3-overexpressing cell lines were analyzed in more detail by mass spectrometry. Subsequent validation of single histone marks in both cells and primary tumor tissue was performed by western blot analysis. RESULTS: In addition to previously identified alterations in gene expression and DNA methylation, we now show that pedGBMs expressing mutant H3.3 display distinct patterns of histone modifications. We demonstrate that the expression of both H3.3 mutants (K27M and G34R/V) induces comprehensive changes in the crosstalk of PTMs. A very striking dominant-negative effect is observed in pedGBMs expressing the K27M mutant, with tumors displaying global downregulation of the key epigenetic mark H3K27me3. Moreover, several PTMs were found to be deregulated in the tail of G34R-mutant H3.3. CONCLUSIONS: Our findings provide novel insights into the complex alterations of the epigenetic code induced by mutant histone H3.3 in pedGBM. We demonstrate global K27M-induced downregulation of the key histone mark H3K27me3, which might results in aberrant gene expression due to altered chromatin structure. In line with this, our results also provide evidence that arginine 34 at the tail of G34R-mutated H3.3 interferes with critical histone marks, leading to aberrant genome output. Taken together, our data provides evidence that at least some of the genome-wide (epi)genetic and transcriptomic alterations found in H3.3-mutant pedGBMs are likely explained by dysregulated histone modifications.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0033. DEVELOPMENT OF A NOVEL SMALL MOLECULE INHIBITOR OF SMOOTHENED FOR MEDULLOBLASTOMA TREATMENT

Minyong Chen 1, Jiuyi Lu 1, Jiangbo Wang 1, Stephen Keir 1, Min Zhang 1, Shengli Zhao 1, Robert Mook 1, Lawrene Barak 1, H Kim Lyerly 1, Wei Chen 1

Abstract

INTRODUCTION: Hedgehog signaling pathway plays a key role for embryo development and adult tissue homeostasis. In brain, Hedgehog signaling controls cerebellum development. Abnormal activation of Hedgehog signaling is one of the important signaling mechanisms contributing to the pathogenesis of medulloblastoma. Increasing evidence has indicated that small molecule inhibitors of the Hedgehog pathway represent an attractive approach in the treatment of medulloblastoma patients. Currently several small molecules are being evaluated in different stages of clinical trials. METHOD: We utilized a high throughput screening drug discovery platform to identify small molecules against Smoothened activity using cells stably expressing beta-arrestin2-GFP and Smoothened receptor. The anti-Smoothed activity of the lead compound was further confirmed by a competition binding assay using radiolabeled cyclopamine, a Gli reporter assay, and a Ptchflox/flox/GFAP-Cre mouse model. RESULTS: We identified a potent Smo inhibitor ZM that directly competed with cyclopamine for binding Smo receptors. As a consequence, the Gli-reporter activity and Gli mRNA expression in PZp53 medulloblatoma cells as well as Hedgehog-dependent proliferation of granule cell precursor cells were significantly inhibited by ZM compound treatment. Interestingly, ZM-mediated inhibitory effect on hedgehog signaling was not affected by the presence of a-acid glycoprotein (AAG), whereas AAG was reported to reduce the efficacy of GDC-0049 in inhibiting Smo activity in vivo. More importantly, oral dosing of ZM led to prolonged survival of Ptchflox/flox/GFAP-Cre medulloblastoma mice. CONCLUSIONS: Taken together, our study identified ZM compound as a potent and effective Smo antagonist that can be further developed as a drug candidate for medulloblastoma treatment.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0034. SYNERGISTIC EFFECT OF SUPERCRITICAL-ETHANOLIC EXTRACTS OF CURCUMA SPECIES WITH ANTICANCER DRUGS IN GLIOMA CELL LINES

Cheppail Ramachandran 1, Smitha Nair 1, Enrique Escalon 1, Ziad Khatib 1, Karl-W Quirrin 2, Steven Melnick 1

Abstract

INTRODUCTION: Brain tumors are one of the deadly forms of human malignancies with a high degree of morbidity and mortality. Pediatric brain tumors are also the most common form of solid tumor in children accounting for about 20-25% of all pediatric cancers. Chemotherapy options for brain tumor treatment are very much limited because of the blood brain barrier and emergence of drug resistance in brain tumor cells. Combining nutraceuticals or botanical drugs with cancer drugs is one of the ways to improve the efficiency of chemotherapy and quality of life in an integrative oncology setting. METHOD: Cytotoxicity of anticancer drugs [Etoposide-ETP, Temozolomide-TMZ) and supercritical-ethanolic (SE) extracts of Curcuma amada (CA-CO), C. xanthorrhiza (CX-CO) and C. longa (CL-CO), curcumin and Turmeric Force either as single agent or their combinations in glioma cell lines (U87MG, U188 MG) were analyzed MTT assay. Synergism, additiveness or antagonism between cancer drugs and SE extracts were determined using CompuSyn analysis of cytotoxicity data. Apoptosis and necrosis induced by different agents or their combinations were analyzed using Roche Annexin-V-FLUOS staining kit. The expression of genes associated with apoptosis and cell proliferation (p53, p21, Bcl-2, Bax, and P10) were determined by RT-PCR assay. RESULTS: Both glioma cell lines are generally resistant to cancer drugs such as TMZ. CA-CO has superior cytotoxic effects as compared to CX-CO and CL-CO. CA-CO also had significantly better cytotoxic effects than curcumin and Turmeric Force. Compusyn analysis of cytotoxic data showed that the combination of ETP and/or TMZ with CA-CO produced synergistic effects on cytotoxicity. The combination of cancer drugs with CA-CO induced higher percentage of apoptosis and necrosis than individual agents. Gene expression studies showed that CA-CO down regulated the expression of P10 and P53 genes and increased the ratio of Bax/Bcl-2 mRNAs. CONCLUSIONS: CA-CO showed synergistic cytotoxicy with cancer drugs such as etoposide and temozolomide in glioma cell lines that is induced by changes in the expression of specific genes affecting cell proliferation and programmed cell death. The combination effect for CA-CO is significantly better than other Curcuma species. The presence of specific chemical constituent difurocumenolol besides turmerones and curcuminoids in CA-CO may be one of the reasons for its superior therapeutic effect than other Curcuma species, curcumin or Turmeric ForceTM. These positive results suggest the need for continuous evaluation of CA-CO in xenograft models and clinical trials in brain tumor patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0035. GENE THERAPY NANOVEHICLE FOR OVERCOMING RADIATION RESISTANCE IN PEDIATRIC BRAIN TUMORS

Forrest Kievit 1, Zachary Stephen 1, Kui Wang 1, John Silber 1, Richard Ellenbogen 1, Miqin Zhang 1

Abstract

INTRODUCTION: Radiotherapy (RT) is an integral component of the treatment for medulloblastoma (MB) and the only effective adjuvant therapy for ependymoma (EP). Survival is frequently accompanied by one or more radiation-induced adverse developmental and psychosocial sequelae, as MB and EP most frequently occur in children less than 10 years old. These considerations emphasize the need to develop new strategies to enhance the tumoricidal action of RT while sparing adjacent normal tissue. The multifunctional DNA repair protein Ape1/Ref-1 has been implicated in conferring radiation resistance in pediatric brain tumors. Our goal is to suppress Ape1 activity through nanoparticle mediated anti-Ape1 siRNA delivery. METHOD: Iron oxide nanoparticle (NPs) were coated various combinations of polymers including chitosan, PEG, and PEI, and tested for DNA binding and protection using size, zeta potential, and electrophoresis analyses. Delivery of green fluorescent protein (GFP) encoding plasmid DNA was tested in vitro and in vivo to model C6 brain cancer cells. Additionally, anti-GFP siRNA was delivered in vitro and in vivo to GFP expressing C6 cells. Chlorotoxin targeted NP delivery to the tumors was verified through magnetic resonance imaging (MRI). GFP fluorescence was monitored to assess successful DNA or siRNA delivery using flow cytometry and fluorescence imaging. RESULTS: NPs coated with a copolymer comprising chitosan, PEG, and PEI (namely, NP-CP-PEI) provided the best combination of high transfection efficiency and low toxicity. NP-CP-PEI provided similar transfection efficiencies as commercially available agents in vitro and were able to transfect brain tumor cells in vivo. Chlorotoxin targeted NPs were able to transfect a higher number of brain tumor cells throughout the tumor in vivo even though the degree of tumor uptake was not affected by targeting as determined by MRI. NPs loaded with siRNA were able to knockdown transgene expression in vitro and in vivo in orthotopic brain tumors. CONCLUSIONS: NP-CP-PEI were shown to be effective at delivering DNA and siRNA both in vitro and in vivo indicating the NPs are able to bypass physiological and cellular barriers to delivery functional DNA and siRNA. Notably, the addition of the targeting agent chlorotoxin to the NPs did not increase the bulk tumor uptake, but did increase the distribution of the NP throughout the tumor to transfect a larger proportion of cells. Therefore, these NPs should be effective at delivering anti-Ape1 siRNA specifically to pediatric brain tumors to increase sensitivity to RT.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0036. TREATMENT OF MEDULLOBLASTOMA WITH MODIFIED MEASLES VIRUS ENCODING AN ENDOSTATIN-ANGIOSTATIN FUSION PROTEIN

Brian Hutzen 2, Adam Studebaker 2, Anna Bratasz 2, Kimerly Powell 2, Corey Raffel 1

Abstract

INTRODUCTION: Measles virus (MV) is effective at treating medulloblastoma in murine xenograft models of intracerebral and CSF-disseminated tumor. Treatment is often, but not always, curative. To increase the efficacy of the virus, we have manufacured two MV that contain either a transgene for human endostatin-angiostatin (hEA) or murine endostatin-angiostatin (mEA). We show that biologically active EA is secreted by cells infected with either virus. We have tested the oncolytic activity of each virus separately and in combination in our intracerebral xenograft model. We find that the combination of MV-hEA and MV-mEA is more efficacious at prolonging survival than either virus alone. METHOD: The hEA or mEA transgenes were inserted between the viral H and L genes. Production of EA by infected cells was measured by ELISA. Supernatents from infected cells were assayed for EA activity by assessing their effect on HUVEC cells treated with VEGF. Intracerebral tumors were established by stereotaxic injection of 106 D283luc cells. Tumors were treated with one intratumoral injection of 2x105 TCID50 of virus 10 days later. Tumor perfusion was measured by MR-based dynamic contrast enhancement (DCE), changes in tumor gene expression was investigated by dot blot, and survival benefit from treatment was determined by the Kaplan-Meyer method. RESULTS: MV-hEA and MV-mEA were effective at killing medulloblastoma cells in vitro. EA secreted by infected cells inhibited proliferation, migration, and tubule formation of VEGF treated HUVEC cells. Unmodifed MV treated tumor-bearing animals survived longer than untreated animals, but the addition of hEA or mEA to the virus did not furhter increase survival. Interestingly, combination treatment with both MV-hEA and MV-mEA (1x105 of each) resulted in the longest survival (p = 0.06). Changes in tumor expression of genes associated with angiogenesis was demonstrated. DCE show little change in tumor perfusion, likely due to the large volume of tumor necrosis observed. CONCLUSIONS: We have demonstrated that the insertion of a human or murine EA transgene into MV results in the secretion of biologically active EA by infected cells. Treatment of tumors with MV-hEA alters expession of angiogenisis-related genes. Paradoxically, little improvement in survival was seen when animals were treated with MV-hEA or MV-mEA compared to unmodified virus. Surprisingly, treatment with the combination of MV-hEA and MV-mEA resulted in better survival than that seen with either EA virus alone. This result suggests that the endothelium in this xenograft model may be derived both from the tumor and from the host.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0037. GLOBAL IDENTIFICATION OF MLL2-TARGETED LOCI REVEALS MLL2'S ROLE IN DIVERSE SIGNALING PATHWAYS

Changcun Guo 1, Chun-Chi Chang 1, Matthew Wortham 1, Lee Chen 1, Dawn Kernagis 1, Xiaoxia Qin 1, Young-wook Cho 2, Jen-Tsan Chi 1, Gerald Grant 1, Roger McLendon 1, Hai Yan 1, Kai Ge 2, Nickolas Papadopoulos 3, Darell Bigner 1, Yiping He 1

Abstract

INTRODUCTION: Myeloid/lymphoid or mixed-lineage leukemia (MLL) family genes encode histone lysine methyltransferases that play important roles in epigenetic regulation of gene transcription. MLL genes are frequently mutated in human cancers. In particular, the MLL2 pathway genes, including MLL2, MLL3, and Utx, have been found to have frequent driver mutations in pediatric medulloblastoma. Unlike MLL1, MLL2 (also known as ALR/MLL4) and its homologue MLL3 are not well understood. Specifically, little is known regarding the extent of global MLL2 involvement in the regulation of gene expression and the mechanism underlying its alterations in driving tumorigenesis. METHOD: Here we used an innovative somatic gene editing-based assay and profiled the global loci targeted by MLL2, and performed a combinatorial analysis of the MLL2 binding profile and gene expression in MLL2 wild-type versus MLL2 null isogenic cell lines. RESULTS: We identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways, including the p53 pathway, cAMP-mediated signaling, and cholestasis signaling. In particular, we demonstrated that MLL2 participates in retinoic acid receptor signaling by promoting retinoic acid-responsive gene transcription. CONCLUSIONS: Our results present the first genome-wide integrative analysis of the MLL2 complex and suggest potential mechanisms underlying the tumorigenesis driven by MLL2 alterations.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0038. LEUKEMIA ASSOCIATED PROTEIN BCL11B IS ASSOCIATED WITH C-MYC IN MEDULLOBLASTOMA AND PROMOTES A GROWTH ADVANTAGE IN VITRO

Brian Cristiano 1, Sujatha Venkataraman 1, Diane K Birks 1, Irina Alimova 1, Peter S Harris 1, Adrian Dubuc 2, Michael D Taylor 2, Nicholas K Foreman 1, Rajeev Vibhakar 1

Abstract

INTRODUCTION: Medulloblastoma are a heterogeneous group of highly malignant brain tumors that most often affect children. Outcomes among children vary widely and C-MYC amplification is the single most important molecular indicator of poor prognosis. Here we analyzed gene expression in high C-MYC expressing tumors and compared these results to low C-MYC expressing controls to identify new therapeutic targets for high C-MYC expressing medulloblastoma. METHOD: We analyzed whole genome mRNA expression in 5 primary tumor samples with high C-MYC expression and anaplastic histology compared to that in 14 low C-MYC expressing tumors with classic histology to identify the leukemia associated zinc-finger protein Bcl11b as upregulated in high C-MYC expressing anaplastic tumors. This observation was externally verified in a dataset of over 103 primary medulloblastoma tumor samples. We used shRNA to examine the impact of BCL11B silencing on cell growth, proliferation and tumor self renewal in vitro. Finally, we utilized immunoprecipitation to identify physical interactions between Bcl11b and C-Myc. RESULTS: Here we show that BCL11B is over expressed in high C-MYC expressing primary tumor samples (p < 6.63E-3), and that targeted disruption of BCL11B by RNAi strongly impairs medulloblastoma cell growth, suppresses tumor cell self-renewal (P < 0.0001, n = 3) and induces apoptosis (p = 0.0458, n = 4). Using immunoprecipitation we also demonstrate a novel physical interaction between C-Myc and Bcl11b. CONCLUSIONS: The negative prognostic value of C-Myc amplification in medulloblastoma has been well established clinically. These results indicate that the leukemia associated zinc finger protein Bcl11b may be an important player in C-Myc driven oncogenesis. Moreover these results demonstrate a physical interaction between C-Myc and Bcl11b that, to our knowledge, has not been previously described. The specific mechanisms of this interaction remain to be elucidated, however it seems clear that BCL11B expression is necessary to support elevated C-Myc protein levels. Thus, Bcl11b or its downstream effectors may represent novel therapeutic targets in high C-MYC expressing medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0039. GENOMIC ANALYSIS OF INTRACRANIAL GERM CELL TUMORS

Koichi Ichimura 1, Shintaro Fukushima 1, Yasushi Totoki 1, Tomonari Suzuki 2, Akitake Mukasa 3, Nobuhito Saito 3, Toshihiro Kumabe 4, Teiji Tominaga 4, Keiichi Kobayashi 5, Motoo Nagane 5, Toshihiko Iuchi 6, Masahiro Mizoguchi 7, Tomio Sasaki 7, Kaoru Tamura 8, Kazuhiro Sugiyama 9, Yoshitaka Narita 1, Soichiro Shibui 1, Masao Matsutani 2, Tatsuhiro Shibata 1, Ryo Nishikawa 2

Abstract

INTRODUCTION: In Japan, intracranial germ cell tumours (iGCTs) are the second most common central nervous system (CNS) tumours in patients under the age of 14. The majority of germinomas respond well to combined chemo- and radiotherapy, however some show resistance to therapy and may have a poor clinical outcome. Despite their clinical significance, the biology of iGCTs is mostly unknown. The aim of the study is to elucidate molecular pathogenesis of iGCTs through a comprehensive genomic analysis. METHOD: In a collective effort to facilitate a biological analysis of CNS GCTs, an Intracranial Germ Cell Tumour Consortium has been formed in Japan. Tumour materials and the patients' clinicopathological information are collected via the Consortium. To study a genetic basis of iGCTs, somatic mutations in all coding exons and the copy number status are investigated by means of whole exome sequencing (WES) and array-comparative genomic hybridization (aCGH) in a selected set of tumours. Statistically significantly mutated genes identified by WES are validated in an independent tumour cohort using the IonTorrent PGM system. RESULTS: A total of 41 centres have so far joined the IGCT Consortium, through which 92 IGCTs (38 germinomas and 54 non-germinomas) have been collected. Of these, 33 tumours were subjected to WES and 56 to aCGH. On average, 29 non-synonymous somatic mutations were observed in each tumour. Mutually exclusive mutations of c-kit and RAS, or amplification of c-kit, were the most common abnormalities, predominantly found in germinomas. In the tumours that do not have c-kit mutations, MTOR, NF1 and EGFR were among the genes mutated. CONCLUSIONS: A comprehensive genomic analysis of CNS GCT indicated that alterations of several receptor tyrosine kinase/MAPK signal transduction pathways play a critical role in the pathogenesis of iGCTs, particularly in germinomas. This finding may lead to the development of a targeted therapy for therapy-resistant iGCTs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0040. FAMILIAL MEDULLOBLASTOMA: USING NEXT-GENERATION SEQUENCING TO UNCOVER PREDISPOSITION IN CLINICALLY SPORADIC MEDULLOBLASTOMA PATIENTS

Paul Northcott 1, Thomas Zichner 2, David Jones 1, Marcel Kool 1, Natalie Jager 1, Maria Feychting 3, Birgitta Lannering 3, Tore Tynes 3, Finn Wesenberg 3, Peter Hauser 4, Young Shin Ra 5, Karel Zitterbart 6, Nada Jabado 7, Jennifer Chan 8, Dan Fults 9, Sabine Mueller 10, Wiesia Grajkowska 11, Peter Lichter 1, Jan Korbel 2, Stefan Pfister 1

Abstract

INTRODUCTION: The recent application of high throughput genomics to large series of clinical medulloblastoma samples has dramatically improved our understanding of key somatic alterations underlying medulloblastoma and its molecular subgroups. Despite this advancement, the contribution of predisposing alleles in the germline of medulloblastoma patients remains largely unexplored. Fewer than 5% of medulloblastomas can be attributed to underlying cancer predisposition syndromes resulting from inherited mutations in just a handful of known cancer genes. Given that 10-15% of clinically sporadic medulloblastoma patients will develop secondary malignancies, the contribution of germline mutations in these individuals may in fact be markedly higher than anticipated. METHOD: As part of the PedBrain initiative within the International Genome Consortium (ICGC), we have performed whole genome sequencing on >150 patient-matched tumour/normal pairs to identify somatic and germline variants causative in medulloblastoma. To increase the power of our study and specifically focus on germline variants, we are currently sequencing exomes of an additional ∼1,000 germlines isolated from medulloblastoma patients and a large series of healthy controls. Variants identified in the germline are filtered against multiple control databases including the 1,000 Genomes Project and the NHLBI Exome Sequencing Project and scored for their predicted functional impact using multiple available bioinformatic tools. RESULTS: Preliminary analysis of germline variants disclosed in our study supports a higher than expected frequency of potentially causative mutations in the medulloblastoma germline. Notable tumour suppressors including PTCH1, SUFU, and TP53 were recognized as recurrently targeted in the germline of this cohort, and importantly, were identified in patients not clinically diagnosed with an underlying predisposition syndrome. Genes not previously reported in the medulloblastoma germline have also been uncovered, including subgroup-enriched candidates. Current efforts are aimed at verifying the overall mutation frequency of these candidates and risk associated with them, as well as their functional relevance as initiating events in medulloblastoma. CONCLUSIONS: Our initial survey of the medulloblastoma germline strongly suggests that predisposing mutations are significantly more common than previously estimated. Ongoing work will continue to investigate the biological, clinical, and ethical implications of our findings.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0041. GENOME SEQUENCING OF SHH MEDULLOBLASTOMA PREDICTS AGE-DEPENDENT RESPONSE TO SMOOTHENED-INHIBITION AND RATIONAL THERAPEUTIC COMBINATIONS

Marcel Kool 1, David TW Jones 1, Natalie Jaeger 1, Paul A Northcott 1, Trevor Pugh 2, Volker Hovestadt 1, Shirley L Markant 3, Lourdes A Esparza 3, Franck Bourdeaut 4, Marc Remke 5, Michael D Taylor 5, Yoon-Jae Cho 6, Scott L Pomeroy 7, Ulrich Schueller 8, Andrey Korshunov 1, Roland Eils 1, Robert J Wechsler-Reya 3, Peter Lichter 1, Stefan M Pfister 1

Abstract

INTRODUCTION: Smoothened (SMO)-inhibitors have recently entered clinical trials for SHH-driven medulloblastoma (SHH-MB). Early evidence suggests that even within this molecularly defined patient subgroup response to therapy is highly variable. To better understand the mechanism(s) of primary resistance to conventional SMO-inhibitors and identify other pathways coopting aberrant SHH signaling, we conducted a comprehensive next-generation study of 125 SHH medulloblastomas obtained from patients between 0 and 49 years of age. METHOD: Tumor and blood DNA of 57 SHH-MBs (31 pediatric [<16 years of age] and 26 adult [≥16]) were subjected to whole-genome or whole-exome sequencing. Two independent non-overlapping replication cohorts (41 pediatric and 27 adult) were sequenced for at least ∼400 prioritized candidate genes. Gene expression (n = 112), DNA copy number (n = 266), and DNA methylation (n = 55) data complement this integrative genomics approach. SHH-MB models with different underlying genotypes were used to evaluate response to SMO inhibition. RESULTS: Three age-related subgroups exhibiting highly discriminate genomic profiles were identified. Mutations in the SHH-pathway involved PTCH1 (young children and adults), SUFU (infants, including germline), and SMO (adults). Children (10–15 years) harbored excess downstream MYCN and GLI2 amplifications, and frequent TP53 mutations, often in the germline, all of which were exceedingly rare in infants and adults. Based on recurrent mutations in other pathways, rational candidates for combination therapies include PI3K-inhibition in infants and adults, GLI-inhibitors in children, and epigenetic modifiers in adults. Functional assays demonstrated that adult SHH-MBs were responsive to SMO inhibition, whereas pediatric counterparts harboring MYCN/GLI2 amplifications were not. CONCLUSIONS: Our data shows that (i) response to SMO inhibition can be predicted by thorough molecular analysis of the primary tumor, (ii) tumor predisposition (Gorlińs Syndrome in infants and Li-Fraumeni-Syndrome in older children) is highly prevalent in patients with SHH-MB, and (iii) rational age group-specific combination therapies may be derived from these findings.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0042. ESTABLISHMENT OF SEVEN NEW PERMANENT PEDIATRIC BRAIN TUMOR LINES

Stephen Keir 1, Charles Pegram 1, Eric Lipp 1, Ahmed Rasheed 1, Vidyalakshmi Chandramohan 1, Chien-Tsun Kuan 1, Madan Kwatra 1, Hai Yan 1, Darell Bigner 1

Abstract

INTRODUCTION: There is an urgent need for clinically relevant cell lines and xenograft models that replicate the biologic phenotypes of human pediatric brain tumors so we can better understand this disease and develop more effective tumor-specific therapies. Current research relies heavily on existing pediatric brain tumor cell lines and xenograft models as a preclinical screen for the development of novel therapies. As the field moves towards treating pediatric brain tumors according to their molecular characteristics, establishing new cell and xenograft lines that represent the genetic diversity of these tumors becomes paramount. Here, we describe 7 new permanent pediatric brain tumor lines. METHOD: All cell and xenograft lines were established from patient-derived tissue at the time of tumor resection. For in vitro establishment, tumor material was washed with PBS, finely minced and digested. From this mixture, a cell-rich supernatant was obtained and washed with zinc option (ZO)-10% fetal bovine serum and treated with Ficoll-Hypaque to remove red blood cells, then washed and cultured in ZO medium. Preparation for in vivo implantation consisted of tumor segmentation with a modified tissue press under sterile conditions. Segmented tissue/homogenate was then loaded into a syringe and injected into the flank of a NOD skid mouse. RESULTS: Seven previously unreported permanent pediatric brain tumor lines were established: 2 glioblastomas, 1 anaplastic ependymoma, 1 atypical teratoid rhaboid tumor, 1 epitheloid chordoma, 1 oligodendroglioma and 1 pilocytic astrocytoma. Of these lines, 3 were established directly as cell culture lines and 3 were established as xenograft lines. Only 1 line grew both as a cell and xenograft line directly from patient-derived tissue. Demographically, 4 lines were established from male patients, 5 lines were from patients with recurrent tissue that had either been treated with radio- or chemotherapy, and 5 of these lines originated from either the frontal or temporal lobe. CONCLUSIONS: As therapeutic advances are made in treating pediatric brain tumors according to their molecular characteristics, additional models, such as the aforementioned, are needed to better represent the genetic diversity that exists within these tumors. It is only through the establishment and maintenance of permanent pediatric cell lines and xenografts that provides researchers with access to a broad collection of representative tumors. Consistent accessibility to these pediatric brain tumor lines aids in both basic and preclinical research as we try to better understand and treat pediatric brain tumors in patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0043. INVESTIGATING THE ROLE OF POLY-(ADP-RIBOSE)-POLYMERASE AS A THERAPEUTIC TARGET IN PEDIATRIC HIGH GRADE GLIOMA

Yevgen Chornenkyy 1, Pawel Buczkowicz 1, Sameer Agnihotri 1, Oren Becher 1, Cynthia Hawkins 1

Abstract

INTRODUCTION: Pediatric supratentorial high-grade astrocytomas (pHGAs) and diffuse intrinsic pontine gliomas (DIPG) are devastating of pediatric malignancies for which no effective therapies exist. Poly-(ADP-Ribose)-Polymerase (PARP) protein expression is found in about 60% of DIPGs and is often accompanied by deficiencies in other DNA repair pathways including BRCA, suggesting PARP may be a potential therapeutic target. While there are multiple ongoing clinical trials utilizing PARP inhibitors for various malignancies there is a lack of pre-clinical data on the potential efficacy of PARP inhibition in pHGA and DIPG. METHOD: Levels of PARP1 and PARP2 were characterized by Western blotting in normal human astrocytes (NHA), pHGA cell lines (SJG2, SF-188), DIPG cell lines (DIPG-M, DIPG58), and one murine brainstem glioma cell line (mBSG). Cell viability in response to different dosages of Olaparib, Veliparib, or Niraparib was determined using the MTT Assay. Reduction of PARP activity and apoptosis was determined by Western blotting against PAR and cleaved PARP, respectively. Gene expression and SNP analysis was used to analyze DNA repair pathways in HGA and DIPG patient cohorts. RESULTS: Western blotting demonstrated that, compared with NHAs, PARP1 and PARP2 were highly expressed in SJG2 and DIPG-M cells. PARP1 expression in mBSG and SF188 was similar to NHA and PARP2 expression was reduced. All PARP inhibitors reduced PARP activity as indicated by reduced PAR levels. Olaparib significantly reduced SJG2 and mBSG cell viability at concentrations of 5uM or 10uM (P < 0.05), while DIPG-M and SF-188 cell viability was reduced only at 10uM (P < 0.05). Veliparib failed to reduce cell viability in all cell lines tested. CONCLUSIONS: Our data provides in vitro evidence that PARP inhibition may be an effective therapeutic avenue for treatment of pHGA and DIPG. Further, it suggests that only cases with high PARP1 and 2 levels may be responsive to this line of therapy emphasizing the need to stratify patients based on PARP expression levels. Furthermore while all PARP inhibitors suppress PARP activity not all PARP inhibitors reduce cell viability. Thus not all PARP inhibitors can be expected to be equally efficacious in a clinical trial setting.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0044. PI3K PATHWAY ACTIVATION PROVIDES A NOVEL THERAPEUTIC TARGET FOR PEDIATRIC EPENDYMOMA AND IS AN INDEPENDENT MARKER OF PROGRESSION FREE SURVIVAL

Hazel Rogers 1, Cerys Mayne 1, John-Paul Kilday 1, Beth Coyle 1, Richard Grundy 1

Abstract

INTRODUCTION: Currently, there are few effective adjuvant therapies for pediatric ependymoma and prognosis remains poor. There is therefore a need to identify candidate genes and pathways against which therapies can be targeted. The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in cancer with a number of drugs targeting the pathway in current clinical trials. PI3Ks transduce signals from growth factors and cytokines resulting in the phosphorylation and activation of AKT which in turn induces changes in cell growth, proliferation and apoptosis. METHOD: PI3K pathway status was analyzed in ependymoma using gene expression data and immunohistochemical analysis of phosphorylated AKT (P-AKT). The effect of the PI3K pathway on cell proliferation was investigated by immunohistochemical analysis of cyclin D1 and Ki67, plus in vitro functional analysis. To identify a potential mechanism of PI3K pathway activation, PTEN protein expression and the mutation status of PIK3CA was investigated. RESULTS: Genes in the pathway displayed significantly higher expression in supratentorial compared to posterior fossa and spinal ependymomas. P-AKT protein expression, indicating pathway activation, was seen in 72% of tumors (n = 169) and P-AKT expression was found to be an independent marker of a poorer progression free survival. A significant association between PI3K pathway activation and cell proliferation was identified suggesting pathway activation was influencing this process. PTEN protein loss was not associated with P-AKT staining and no mutations were identified in PIK3CA. CONCLUSIONS: Our results suggest the PI3K pathway could act as a biomarker, not only identifying patients with a worse prognosis, but also those which could be treated with therapies targeted against the pathway, a strategy potentially effective in a high percentage of ependymoma patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0045. CELL-INTRINSIC SEX DIFFERENCES UNDERLIE THE MALE PREVALENCE IN BRAIN TUMOR RATES

Tao Sun 1, Nicole Warrington 1, Jingqin Luo 1, Michael Brooks 1, Sonika Dahiya 1, Rajarshi Sengupta 1, Joshua Rubin 1

Abstract

INTRODUCTION: Clinical studies have shown that brain cancers including Glioblastoma Multiforme (GBM), occur more frequently in males than in females worldwide, while the reason for this sexual disparity is poorly understood. More interestingly, the sex difference in the brain tumor rates also exists in pediatric population, in which the brain tumor incidence peaks at about 4 years of age, suggesting that besides the effects of sex hormones, cell-intrinsic sex differences may play a role in determining the sexual dimorphism in the oncogenesis of brain tumors. The goal of the current study is to examine the potential role of sex in gliomagenesis. METHOD: To model the impact of sex on the oncogenesis of glioblastoma, we established a step-wise transformation system for malignant glioma formation by using murine astrocytes with ablation of tumor suppressor, neurofibromin (Nf1). The Nf1-/- astrocytes were further challenged by losing p53 function through overexpressing a dominant-negative p53 (DNp53) molecule. These Nf1-/-;DNp53 astrocytes were either treated with epidermal growth factor (EGF) in vitro, or implanted into immunodeficient mice. In addition, Illumina microarray assays were performed to compare the gene expression profiles in male and female astrocytes in each step of the malignant transformation. RESULTS: EGF treatment results in in vitro transformation of Nf1-/-;DNp53 astrocytes but only in male cells, and this result was further supported by limiting dilution analyses. Consistent with the in vitro data, subcutaneous implants of Nf1-/-;DN-p53 cells into nude mice led to a sexually dimorphic tumor growth pattern. Survival analyses on the nude mice with intracranial implants of the Nf1-/-;DNp53 cells demonstrated a sex-dependent survival pattern with100% death in mice bearing male Nf1-/-;DNp53 cells vs. 41% death in mice carrying the female counterparts. Lastly, microarray analyses revealed an enhanced sex difference in gene expression as a function of oncogenesis. CONCLUSIONS: We established a model system that permits us to model the impact of sex on gliomagenesis. By employing Nf1-/-;DNp53 mouse astrocytes, we were able to demonstrate that male astrocytes are more susceptible to malignant transformation both in vitro and in vivo. Furthermore, our in vivo tumor formation data suggest that it is the cell-intrinsic sex differences rather than circulating sex hormones that may determine the sexual disparity in glioma rate in patients. Lastly, our microarray analyses on gene expression profiles in male and female astrocytes revealed a potential interaction between sex and oncogenic progression during glioma formation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0046. PID1 (NYGGF4), A NEW TUMOR SUPPRESSOR-LIKE GENE IN PEDIATRIC AND ADULT BRAIN TUMORS

Anat Erdreich-Epstein 1, Nathan Robison 1, Xiuhai Ren 1, Hong Zhou 1, Lingyun Ji 1, Ashely Margo 1, David Jones 2, Stefan Pfister 2, Marcel Kool 2, Richard Sposto 1, Shahab Asgharzadeh 1

Abstract

INTRODUCTION: Medulloblastomas are the most common malignant brain tumors in children, glioblastomas are the most common malignant primary brain tumors in adults, and atypical teratoid rhabdoid tumors (ATRT) are a highly malignant brain tumor in children. PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) is a recently-identified gene that acts as a modulator of insulin signaling in adipocytes and muscle cells, and to date, has only been reported in the context of obesity, diabetes and Alzheimer's Disease. Expression and roles of PID1 in cancer and in brain tumors are still unknown, and are a central topic in our laboratory. METHOD: PID1 mRNA expression in medulloblastoma tumors was assessed by quantitative RT-PCR in the CHLA cohort and from expression microarrays of three published and unpublished independent medulloblastoma datasets (total n = 821). PID1 mRNA level in gliomas was obtained through analysis of online data from REMBRANDT (344 cases) and TCGA (424 cases). In vitro experiments used D283MED (medulloblastoma), U251 and LN229 (glioblastoma), and CHLA-06-ATRT (atypical teratoid rhabdoid tumor) cell lines. Proliferation and cell death were measured by 7AAD/BrdU and annexinV staining. Mitochondrial polarization was assessed using JC-1. RESULTS: PID1 mRNA was lower in clinically-unfavorable medulloblastomas (Groups 3 and 4 or anaplastic histology) compared with clinically-favorable medulloblastomas (SHH and WNT groups or desmoplastic histology) or fetal cerebellum. PID1 mRNA level was also lower in glioblastomas compared to non-tumor brain, astrocytomas and/or oligodendrogliomas. In medulloblastomas higher PID1 mRNA correlated with longer overall survival (OS) and longer radiation-free PFS. Higher PID1 mRNA also correlated with longer OS in gliomas. Ectopic expression of PID1 decreased colony formation, increased cell death, inhibited proliferation, and induced mitochondrial depolarization. Additional new studies on the mechanism of the brain-tumor inhibitory action of PID1 will be presented. CONCLUSIONS: These data are the first to link PID1 to cancer in general and to pediatric and adult brain tumors specifically, and suggest that PID1 has a tumor-inhibitory/tumor-suppressor-like function in medulloblastomas, glioblastomas and ATRTs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0047. BIOMARKER-DRIVEN STRATIFICATION OF DISEASE-RISK IN NON-METASTATIC MEDULLOBLASTOMA: THE SIOP-EUROPE PNET4 CLINICAL TRIAL

Steven Clifford 1, Goran Gustafsson 2, David Ellison 3, Dominique Figarella-Branger 4, Francois Doz 5, Stefan Rutkowski 6, Birgitta Lannering 7, Torsten Pietsch 8

Abstract

INTRODUCTION: The accurate stratification of disease-risk, and delivery of risk-adapted therapies, are major goals in medulloblastoma. Patients aged <3 years and/or with metastatic disease at diagnosis have been considered ‘high-risk’, and their exclusion defined the ‘standard-risk’ group treated on the recent SIOP-PNET4 trial. A series of validated molecular and pathological prognostic markers have now been identified for medulloblastoma. However, whilst favourable-risk WNT subgroup patients will receive reduced therapy in the forthcoming PNET5 clinical trial, risk-biomarkers within the remaining ‘standard-risk’ group are less well defined. METHOD: We undertook a prospective study of biomarkers of reported biological or prognostic significance, alongside clinical and pathological variables, within the PNET4 cohort. Formalin-fixed paraffin-embedded (FFPE) tumour tissue was collected for centralised pathology review (n = 338) and analysis (up to n = 236) of WNT subgroup status (β-catenin immunohistochemistry, CTNNB1 mutation)) and copy-number alterations (chromosome 17, MYC/MYCN, PTCH1 and DNA ploidy status). RESULTS: Five-year event-free survivals with standard (78 ± 3%) and hyperfractionated (79 ± 4%) radiotherapy were equivalent (p = 0.82). β-catenin nuclear positivity predicted a favourable outcome (p = 0.05), while chromosome 17 defects and residual tumour post-surgery were each associated with poor prognosis (p < 0.01). Chromosome 17p/q defects predicted poor prognosis when observed against a diploid, but not polyploid, genetic background. All three factors were independent markers in multivariate testing. Notably, previously identified poor prognosis markers (MYC/MYCN amplification, LCA disease) were not associated with adverse outcome. In survival models, chromosome 17p/q defects predicted poor outcome (56 ± 12%), while cases without 17p/q defects did not diverge significantly from the WNT subgroup. CONCLUSIONS: These findings demonstrate assessment of disease-risk in ‘standard-risk’ 132medulloblastoma requires distinct biomarkers and stratification schemes. The routine testing of chromosome 17 and WNT subgroup status provides a strong basis for improved patient stratification in future clinical trials.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0048. CLINICOPATHOLOGICAL AND GENETIC CHARACTERIZATION OF PEDIATRIC EPITHELIOID GLIOBLASTOMA

Alberto Broniscer 1, Ruth Tatevossian 1, Noah Sabin 1, Paul Klimo Jr 2, James Dalton 1, Ryan Lee 1, Amar Gajjar 1, David Ellison 1

Abstract

INTRODUCTION: The epithelioid and rhabdoid variants of glioblastoma are rare. Although similar in standard histological preparations, they are distinguished by their immunophenotypes. In particular, the rhabdoid glioblastoma (r-GB) contains foci of cells that are immunopositive for multiple diverse proteins and immunonegative for INI1, simulating the phenotype of an atypical teratoid/rhabdoid tumor (AT/RT). In contrast, cells with an epithelioid or rhabdoid morphology in the epithelioid glioblastoma (e-GB) express INI1 and lack the diverse immunophenotype of the r-GB. Here, we report a series of seven pediatric e-GBs describing their clinical, pathological, and genetic characteristics. METHOD: We retrospectively reviewed the clinical characteristics of all patients with epithelioid glioblastoma seen at our institution between 1999 and 2012. Central pathologic and radiologic review of all cases was performed. Molecular genetic and cytogenetic analyses were undertaken, including evaluation of INI1 status and sequencing of mutational hotspots in H3F3A, HIST1H3B, BRAF, IDH1, and IDH2. RESULTS: Seven patients were identified. Median age at diagnosis was 10.2 years. Tumor sites were diencephalon (n = 4) and cerebral cortex (n = 3). Three patients presented with acute, symptomatic intra-tumoral and intra-ventricular hemorrhage. Three patients had leptomeningeal tumor spread at diagnosis. Despite the use of multi-modality therapy, all patients experienced rapid disease progression. First tumor progression for five patients was metastatic, either in the leptomeninges (n = 3) or at extra-axial sites (n = 2). All patients eventually experienced distant tumor progression. Diffuse extra-axial spread was documented at autopsy in two additional cases. Median survival was 5 months (range, 1.4 to 9.7 months). Pathologically, tumors had a dominant epithelioid morphology, and their cells expressed INI1, but not the diverse range of proteins characteristic of AT/RT. One thalamic tumor had an H3F3A:p.K27M mutation. Four tumors (57%; 2 thalamic and 2 cortical) harbored a BRAF:p.V600E mutation. Two tumors showed homozygous loss of CDKN2A, and one tumor harbored EGFR amplification. CONCLUSIONS: We report the first pediatric series of the rare e-GB, which is a particularly aggressive variant with a high frequency of BRAF:p.V600E mutation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0049. MEMBRANE DEPOLARIZATION IS A MEDULLOBLASTOMA TUMOR SUPPRESSOR

Livia Garzia 1, Adrian Dubuc 1, Graham Pitcher 2, Paul Northcott 3, Adrian Mariampillai 4, Tiffany Chan 1, Patryk Skowron 1, Xiaochong Wu 1, Yuan Yao 1, Cynthia Hawkins 1, John Peacock 1, Kory Zayne 1, Sidney Croul 1, James Rutka 1, Anna Kenney 7, Annie Huang 1, Victor Yang 4, Stephen Baylin 13, Michael Salter 2, Michael Taylor 1

Abstract

INTRODUCTION: Extensive studies of MB genomics revealed that only a few genetic events are highly recurrent, and the mutated genes do not appear to converge on well known signalling pathways or biological processes, and do not immediately suggest pathways or targets that could be used to treat a meaningful percentage of patients. Therefore we undertook a genome-wide investigation of medulloblastoma DNA hypermethylation in an effort to determine the genes and pathways driving tumor initiation and progression. METHOD: To discover the epigenetic events driving medulloblastoma pathogenesis, we treated cell lines with a DNA demethylating agent and a histone deacetylase, performed MeDIP-ChIP on both medulloblastomas and normal human cerebella and narrowed the list to genes that were transcriptionally silenced in primary medulloblastomas. We identified candidates that showed tumor specific silencing secondary to CpG hypermethylation, we also examined somatic copy number aberrations (SCNAs) from 122 medulloblastomas. Pathway analysis revealed that genetic and epigenetic events converge on synaptic electrical activity and neuronal membrane homeostasis. We performed pharmacological and optogenetic modulation of membrane potential to functionally asses its role in medulloblastoma. RESULTS: We identified neuronal development as a frequent target of gene silencing. Genes involved in synaptic biology, ion channels and neurotransmitters are also frequently and focally deleted in medulloblastoma. Sonic Hedgehog medulloblastomas arise from cerebellar progenitor cells that give rise to mature glutamatergic neurons. We treated medulloblastoma cultures with glutamate agonists finding a decrease in cellular proliferation and increased differentiation. Using a new optogenetic mouse model of medulloblastoma, we depolarized MB cells using a blue laser. Our optogenetic results cleanly demonstrate that membrane depolarization per se is tumor suppressive as depolarized cells are unable to transplant the disease in immuncompromised mice. Conclusions: Membrane depolarization is targeted by recurrent, highly convergent somatic genetic and epigenetic events in medulloblastoma. Our optogenetic approach provides high level evidence that medulloblastoma cells evade differentiation in response to membrane depolarization through selection of clones with somatic genetic or epigenetic events affecting genes involved in synaptic biology. This selection for cells impaired in synaptic function attenuates membrane depolarization in response to local neurotransmitters, maintains an undifferentiated state, and contributes to tumor malignancy. Membrane depolarization therefore represents a novel form of non-genic tumor suppressor and represents an attractive therapeutic avenue for the treatment of children with malignant brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0050. SONIC HEDGEHOG ACTIVATION MODULATES CXCR7 EXPRESSION AND PROMOTES CXCR4 SIGNALING IN MEDULLOBLASTOMA

Stacey Ward 1, Rajarshi Sengupta 1, Joshua Rubin 1

Abstract

INTRODUCTION: Stratification of medullolastoma into molecular subtypes elevates hopes that subtype-specific therapy can be more efficacious and less toxic. To realize this goal, the molecular targets that distinguish each subtype (more efficacious) and best discriminate between normal and tumor cells (less toxic) must be fully defined. We published that Sonic hedgehog (Shh) subtype medulloblastoma can be further divided by CXCR4 expression into clinically important subgroups and that CXCR4 inhibition has potent anti-tumor effects. Here we present data that Shh regulates CXCR4 activity through CXCR7, whose differential expression between normal granule cells and medulloblastoma cells makes its function an ideal therapeutic target. METHOD: Cells were isolated from P6 mouse cerebellae or from primary mouse cerebellar tumors (SmoA1). Single-cell suspensions were plated on poly-D-lysine coated dishes with or without: 1) Shh and 2) cyclopamine, followed by treatment with CXCL12. SmoA1 brain tumors were dissociated into single cells and transplanted into nude recipient mice, and after growth to 100mm2 were treated with vehicle, AMD3100, GDC-0449, or both. Tumors were measured by digital caliper and harvested when one dimension reached 20mm or at the conclusion of treatment. RESULTS: Using primary cultures of Shh-driven medulloblastoma we found that Shh activation enhances CXCR4 surface localization and promotes CXCL12-induced GαI signaling. This was similar to effects of Shh on cerebellar granule precursor cells, the normal developmental counterpart of Shh-medulloblastoma. We evaluated expression of CXCR4 internalization regulators and found that Shh activation altered the abundance of a subset of GRKs, arrestins and CXCR7. Among these, CXCR7 expression exhibited the greatest differential response to Shh pathway activation between normal granule cells and primary medulloblastoma samples: CXCR7 expression was highly expressed in normal cells despite Shh effects, but almost completely absent in tumor cells. CONCLUSIONS: We conclude that activation of the Shh pathway in medulloblastomas promotes CXCR4 surface localization through inhibition of internalization mediators, which promotes prolonged CXCR4 signaling. This cross-talk is also evident in developing GPCs but with one notable difference: CXCR7 expression is high in Shh-stimulated GPCs and negligible in Smoothened-mutated SmoA1 cells. This distinction suggests that CXCR7 antagonists, which are moving towards clinical trials in a number of cancers, may be detrimental to normal cerebellar development and inactive against medulloblastoma. We propose instead that restoring CXCR7 activity would have negligible effects on granule cells and potently block CXCR4 function in medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0051. RECURRENT MEDULLOBLASTOMA IS HIGHLY DISTINCT FROM ITS MATCHED PRIMARY TUMOR

Livia Garzia 1, Sorana Morrissy 1, Patryk Skowron 1, Salomeh Jelveh 3, Patricia Lindsay 3, David Largaespada 4, Lara Collier 5, Adam Dupuy 6, Richard Hill 2, Michael Taylor 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common paediatric malignant brain tumor. By the way of optimal surgery, radiation, and chemotherapy, medulloblastoma can be treated in a good fraction of children but despite the best therapy the disease recurs in 40% of the cases. Many factors composing the tumor niche can also influence radiosensitivity. For this reason we developed a protocol to study the genetic differences between primary and recurrent (radioresistant) tumors in vivo, using our transposon mutagenesis driven mouse model. METHOD: We developed a novel murine model of metastatic MB, which is highly penetrant, has a short latency, and involves random secondary genetic events. The model is based on mobilizing the Sleeping Beauty transposon in the cerebella of Ptch+/- mice. We used this model to identify the genes important for recurrence of MB in vivo. We performed surgical removal of the murine tumors, and then treated the mice by multi-fraction CT-guided craniospinal irradiation. By the way of next generation sequencing followed by gCIS prediction or convergence analysis we identified mutated driver genes in the primary tumors as compared to the recurrences. RESULTS: 70% of the mice treated with surgery and CSI recurred locally, a smaller fraction (30%) recurred distally with recurrent disease on the spinal cord. Recurrences are highly genetic divergent from their matched primary tumor. We have sequenced the first batch of recurrent mice, comparing primary, local and distal recurrences identifying several potential synthetic lethal genes in the primary tumors, and relapse drivers, which are only found mutated in the recurrences. We selected actionable targets and performed in vitro radiosensitization assays with small molecules inhibitors of the predicted driver genes, showing a reversal of radiation resistance. CONCLUSIONS: Recurrent medulloblastoma can be modelled in mice by the way of surgery followed by CSI irradiation. Murine primary MBs are highly genetically different from the recurrences, urging the scientific community to develop different therapeutic approaches to efficiently target primary and recurrent human tumors. As our mouse model shows the same rate and pattern of recurrence observed in human patients is an extremely valuable translational platform to design new strategy against recurrent MB. Highly targetable events in genes known to play a role in cell-cycle, apoptosis and proliferation, like Trp53 and ALK, are potential drivers of local and distal MB recurrence.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0052. LONGITUDINAL ASSESSMENT OF CIRCULATING microRNAs As Biomarkers In Pediatric Central Nervous System Tumors

Rishi R Lulla 1, Joseph Laskowski 1, Jason Fangusaro 1, Arthur J DiPatri 3, Tord Alden 3, Elio F Vanin 2, Tadanori Tomita 3, Stewart Goldman 1, Marcelo Bento Soares 2

Abstract

INTRODUCTION: Altered expression of miRNAs contributes to the development of cancer including pediatric central nervous system (CNS) tumors. MicroRNAs have tissue specific expression patterns and are stable molecules making them ideal cancer biomarker candidates. Circulating miRNAs have been previously identified in plasma and cerebrospinal fluid (CSF) of pediatric patients with CNS tumors. We determined miRNA expression levels in corresponding tumor, plasma, urine and CSF of newly diagnosed patients with CNS tumors at the time of diagnosis and at routine intervals during their treatment. METHOD: Tumor tissue and body fluids were prospectively collected at specified time points. RNA purification from cell-free body fluids was performed with the miRCURYTM RNA isolation kit and from tumor tissue using RecoverAllTM RNA isolation kit. The purified total RNA was then used for cDNA synthesis. TaqMan® miRNA assays were used to quantify the levels of 762 mature miRNAs from each sample using the Applied Biosystems 7900HT Fast Real-Time PCR system in 384-well low density arrays (TLDAs). Cycle threshold (Ct) values under 33 were considered positive for the presence of target miRNAs. ExpressionSuite® software was used for data analysis. RESULTS: To date, 25 patients are enrolled on the study with longitudinal specimens available for 14 patients. Profiling on 16 patients has been completed. In all cases, a number of miRNAs are present in the tumor and body fluids at the time of diagnosis. For patients with longitudinal samples, several candidate biomarker miRNAS have been identified in patient samples at diagnosis which are no longer detectable in subsequent specimens following initial treatment. For example, in patients with embryonal tumors, miR-30a-5p and miR-661 are candidate biomarkers in the CSF. Profiling of the remainder of the samples is ongoing and will be presented. CONCLUSIONS: Our results establish that circulating miRNAs in plasma, CSF and urine are candidate biomarkers in pediatric CNS tumors. Individual miRNAs of interest vary based upon the histologic subtype of the tumor and expression levels decreased after surgery and/or other tumor directed therapy. Ongoing studies include: continued surveillance of body fluids of patients enrolled on this study and comparison of miRNA profiles of affected patients to those without CNS tumors. We hypothesize that circulating miRNAs may represent a novel diagnostic tool for patients with CNS tumors and may be a valuable biomarker for response to therapy and early indication of relapse.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0053. LABEL FREE PROTEOME PROFILING OF CEREBROSPINAL FLUID IDENTIFIES PROFILIN-1 AS A PUTATIVE BIOMARKER OF MEDULLOBLASTOMA WITH A PRO-MIGRATORY FUNCTION

Meena U Rajagopal 1, Ling San Lau 1, Yetrib Hathout 2, Heather Gordish-Dressman 2, Brian Rood 1

Abstract

INTRODUCTION: A major challenge to the treatment of medulloblastoma is the toxicity of craniospinal radiation to a child's developing brain. Current efforts to risk stratify medulloblastoma therapy to minimize craniospinal radiation and its sequelae would benefit greatly from the ability to assess disease status using serially accessible biomarkers. Unlike brain tumor tissue, cerebrospinal fluid (CSF) represents a reservoir of brain-associated proteins that can be serially sampled. We have identified profilin-1 as a candidate protein biomarker with an important functional role in medulloblastoma biology. METHOD: We used mass spectrometry based label-free protein quantitation (MS-LFPQ) to compare the CSF proteome of 12 children newly diagnosed with medulloblastoma to 12 tumor-free, age-matched controls. Cell lysates and conditioned media from medulloblastoma cell lines were also analyzed by MS-LFPQ. Pathway analysis revealed a significant number of proteins involved in cell motility, including profilin-1, an actin-binding protein implicated in the migration of breast cancer and bladder cancer cells. Profilin-1 expression was validated by ELISA and immunohistochemistry. Migration of medulloblastoma cell lines DAOY and D556 was assessed by transwell migration and wound healing assays following siRNA knockdown of profilin-1 gene expression. RESULTS: We identified 39 proteins that were differentially abundant in CSF from children newly diagnosed with medulloblastoma compared to controls (2.5 fold, p < 0.05), 13 of which were significantly upregulated. Profilin-1 was 11.7 fold elevated in tumor samples (p < 0.03) and detected in 7 out of 12 medulloblastoma samples. We validated increased profilin-1 in medulloblastoma-associated CSF using ELISA. We also found profilin-1 to be abundantly expressed and secreted by medulloblastoma cell lines and present in 59% (13/22) of medulloblastoma cases by immunohistochemistry. In vitro migration assays demonstrated significantly decreased migration in medulloblastoma cell lines following siRNA knockdown of profilin-1. CONCLUSIONS: Our proteomic investigation of CSF from children with medulloblastoma has identified profilin-1 as a promising putative biomarker for medulloblastoma that is detectable in CSF and robustly expressed by tumor tissue and cell lines. We also demonstrate that profilin-1 is involved in the migration of medulloblastoma cells; an important function in tumor biology related to tumor progression and metastasis. Biomarkers with the ability to reflect the biologic characteristics of individual tumors would offer an additional capability to not only assess the presence of disease but provide real-time evidence of in situ tumor biology.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0054. STUDY OF HISTONE METHYL TRANSFERASE G9a INHIBITION IN ATRT AND MEDULLOBLASTOMA

Vrushali Datar 1, Sunil Bochare 1, Akansha Singh 1, S Khatau 1, J Fangusaro 2, S Goldman 2, R Lulla 2, Veena Rajaram 3, Vidya Gopalakrishnan 1

Abstract

INTRODUCTION: Medulloblastoma and atypical teratoid rhabdoid tumor (ATRT) are malignant pediatric brain tumors. The survival rate for children with AT/RT who are below the age of three is around 10%, whereas that for medulloblastoma patients is approximately 75–80%. Survivors face increased risk for recurrence and current therapeutics are ineffective against recurrent tumors. Thus, there is a dire need for novel therapies. Aberrant epigenetic silencing of gene expression has been increasingly implicated in cancer development. One such repressive epigenetic modification is histone H3 lysine (K)-9 methylation, which is catalyzed by histone methyl transferases (HMTs) called G9a and G9a-like protein (GLP). METHOD: A panel of established and patient derived human medulloblastoma cell lines ATRT cell lines were used to measure G9a expression across by q-RT-PCR analyses. Cell growth in response to drug treatment was measured by (MTT) assay. Cell cycle analysis was performed by flow cytometry. Western blot analysis was done to observe global changes in H3K9Me2 following G9a inhibition. In vivo tumorigenic potential of drug treated cells was measured in mouse orthotopic models. Tumors were analyzed by H&E staining and immunohistochemistry. RESULTS: We observed elevated G9a expression in ATRT and human medulloblastoma samples compared to normal cerebellum. Ablation of G9a activity by treatment with small molecule inhibitors of histone methyl transferases decreased ATRT and medulloblastoma cell growth in vitro and in vivo. HMT inhibitor-loss of cell growth in vitro was accompanied by a significant decrease in global histone H3K9 methylation. In vivo, HMT inhibitors decreased the tumorigenic potential of ATRT and medulloblastoma cell lines and surprisingly supported the formation of circumscribed and less invasive tumors. CONCLUSIONS: Treatment of ATRT and advanced stage/relapsed medulloblastoma continues to remain a challenge. This calls for the development of novel therapeutic approaches based on a better understanding of tumor biology. Here, we provide the first demonstration of aberrant expression of G9a in human medulloblastoma and ATRTs. Our data show that pharmacological inhibition of G9a activity blocked tumor cell growth in vitro and in vivo, suggesting that G9a inhibition may have future therapeutic application for pediatric brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0055. HIGH THROUGHPUT SCREEN IDENTIFIES TWO FDA DRUGS THAT SUPPRESS NON-SHH AND NON-WNT MEDULLOBLASTOMA PROLIFERATION

Marie Morfouace 1, Anang Shelat 1, Megan Jaccus 1, Burgess Freeman 1, Frederique Zindy 1, Giles Robinson 1, Kiplin Guy 1, Clinton Stewart 1, Amar Gajjar 1, Martine Roussel 1

Abstract

INTRODUCTION: Medulloblastomas, the most common malignant pediatric brain tumors, are molecularly divided into 4 major groups: SHH, WNT, Group 3 and Group 4. Group 3 tumors, which overexpress and frequently amplify MYC, are the most aggressive and least curable. Our recently described mouse model recapitulates Group 3 medulloblastoma by overexpressing Myc in Trp53-null granule neural progenitors. Myc-induced mouse medulloblastoma cells can be grown as neurospheres and recapitulate primary tumors after transplantation into the brain of nude recipient mice. These properties provide an ideal platform on which to conduct high throughput screens and then test the “hits” in the live model system. METHOD: A library of 830 FDA-approved drugs was screened against Myc-induced mouse medulloblastoma neurospheres (hereon Myc-neurospheres) by measuring ATP consumption. Drugs active against Myc-neurospheres (primary screen, single point at 10 µM) were moved to a secondary screen (dose response from 1 nm to 10 µM) in order to assess their selectivity. Wash out experiments were performed on 21 compounds. Drugs that efficiently inhibited Myc-neurosphere proliferation and that were selective (i.e., inactive against Trp53-null neurospheres) were moved forward and subjected to pharmacokinetic and in vivo studies. RESULTS: Pemetrexed (an inhibitor of the folate pathway) and gemcitabine (an inhibitor of nucleotide synthesis), were active and selective against the Myc-neurospheres. Pharmacokinetic modeling of these compounds demonstrated they were detectable in tumor extracellular fluid at concentrations that exceeded the in vitro IC50 values for 6 hours. When administered as single agents to mice orthotopically transplanted with Myc-neurospheres, each increased the survival by 6 and 10 days, respectively. However, when administered in combination survival doubled from 15 to 30 days. Importantly, they also inhibited the proliferation of several primary neurosphere lines derived from non-SHH/non-WNT human medulloblastomas. CONCLUSIONS: Group 3 medulloblastomas grow robustly as neurospheres allowing for the unbiased high throughput screening of vast drug libraries. Using a library of FDA-approved drugs and preclinical studies we have identified pemetrexed and gemcitabine as candidate drugs for the treatment of patients with this most aggressive form of medulloblastoma. This work is funded in part by The V-foundation (MFR, AG), NIH Grants CA-096832 (MFR), a Core Grant CA-021765and the American Lebanese-Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0056. T CELLS EXPRESSING IL13Rα2-SPECIFIC CHIMERIC ANTIGEN RECEPTORS BASED ON IL13.E13K MUTEINS HAVE POTENT ANTI-GLIOMA ACTIVITY BUT ALSO RECOGNIZE IL13Rα1+ CELLS

Simone Krebs 1, Kevin Chow 1, Zhongzhen Yi 1, Vita Brawley 1, Nabil Ahmed 1, Stephen Gottschalk 1

Abstract

INTRODUCTION: New targeted therapies are needed for pediatric patients with high grade gliomas, and adoptive T-cell therapy has the potential to fulfill this need. One attractive target antigen for T-cell immunotherapy is IL13Rα2, which is expressed in >50% of pediatric high grade gliomas. While two groups have reported the construction of IL13Rα2-specific chimeric antigen receptor (IL13Rα2-CARs) using IL13 muteins, at present it is unclear, which IL-13 mutein is optimal for CAR generation. The goal of this project was to evaluate different IL13 muteins for CAR generation and determine the effector function of IL13Rα2-CAR expressing T cells. METHOD: We constructed four SFG retroviral vectors encoding IL13Rα2-CARs. While the IL13Rα2-CARs had identical IgG1-CH2CH3 spacers, CD28 transmembrane domains, and signaling domains derived from CD28 and the CD3-ζ chain, their extracellular binding domain contained IL13 muteins, with increased affinity to IL13Rα2. Two had a single amino acid substitution at position 13 (E13K and E13Y), and two had an additional amino acid substitution at position 105 (K105R). CAR-expressing T cells were generated by retroviral transduction, and the phenotype, specificity, and antitumor activity of CAR T cells was determined using standard immunological assays. RESULTS: All four IL13Rα2-CARs were expressed at >80% on the cell surface of transduced T cells. IL13Rα2-CAR T cells were activated in the presence of IL13Rα1 or IL13Rα2 protein in contrast to control protein (IL4R) as judged by cytokine production. In cytotoxcity assays IL13Rα2-CAR T cells killed L13Rα1+ and/or IL13Rα2+ target cells in contrast to target cells that were negative for IL13Rα1 and IL13Rα2. While we observed no significant differences between the four constructed IL13Rα2-CARs ex vivo, only T cells expressing CARs with E13K amino acid substitutions (IL13.E13K and IL13.E13K;K105R) induced regression of established gliomas in the U373 orthotopic xenograft model. CONCLUSIONS: T cells expressing IL13Rα2-CARs with IL13.E13K muteins have significant antitumor activity in vivo. However, IL13 mutein-based CARs also recognize IL13Rα1, which will limit the use of IL13 mutein-based CAR T cells to local applications. Our results provide the rationale to develop IL13Rα2-specific CARs that use an IL13-specific single variable fragment as an antigen recognition domain for IL13Rα2 to prevent cross reactivity to IL13Rα1.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0057. ANALYSIS OF CELLULAR AND MOLECULAR CHANGES ACCOMPANYING BRafV600E-TARGETED TREATMENT IN A MODEL OF PEDIATRIC ASTROCYTOMA

Robin Lerner 1, Julie Harness 1, Yasuyuki Yoshida 1, Raquel Santos 1, Jacqueline De La Torre 1, Theo Nicolaides 1, Tomoko Ozawa 1, David James 1, Claudia Petritsch 1

Abstract

INTRODUCTION: In a subset of pediatric malignant astrocytoma (MA), activating BRafT1799A mutation leads to expression of hyper-activated BRafV600E kinase, this frequently occurs alongside deletion of the cell cycle inhibitor Cdkn2a (Cancer Res. 70:512-9; 2010). BRafV600E-targeted therapies retard the growth of intracranial BRafV600E MA xenografts (Clin. Cancer Res. 17:7595-604; 2011). Stem and progenitor-like glioma cell subpopulations have been shown to exhibit differential therapy responses, and consequently contribute in distinct manners to therapy resistance (Cancer Cell. 18:669-82; 2010). The potentially divergent effects of BRafV600E -targeted therapies on heterogenous MA cell subpopulations have not been previously explored. METHOD: Athymic mice were orthotopically implanted with AM38, a human BRafV600E and Cdkn2a null MA cell line, modified with lentivirus-luciferase. Tumor-bearing mice were treated with BRafV600E-specific inhibitor PLX4720, or left untreated. Whole brains were harvested and subjected to immunohistopathological analyses; tumor cells were isolated and analyzed by flow cytometry. Stem (CD133) and progenitor (NG2) cell markers, co-labeling with BrdU, were used to identify and quantify proliferation rates and rates of asymmetric divisions of distinct cancer stem-like cells subpopulations. Tumor tissue and AM38 cells at drug-responsive and un-responsive stages were comparatively analyzed. RESULTS: AM38 cells injected into athymic mouse brain produce astrocytoma-like tumors, with animal hosts quickly succumbing to tumor burden. Treatment with PLX4720 retards tumor growth and extends survival. Untreated and PLX4720-treated tumors harbor distinct proportions of MA subpopulations marked by CD133+and NG2+ expression, and having distinct proliferation capacities in situ. Rates of asymmetric divisions of CD133+ and NG2+ cells were determined. Results from ongoing work for further analysis of treatment effects on tumor stem and progenitor subpopulation composition in situ, as well as on subpopulation proliferation and asymmetric division, in vitro, will be presented at the meeting. CONCLUSIONS: Our studies show multiple cell subpopulations in AM38 cultures and orthotopic xenografts. PLX4720 retards AM38 orthotopic xenograft growth, and in so doing may alter tumor cell subpopulation composition. Ongoing work will explain the impact of this BRafV600E inhibitor effect, especially with respect to CD133+ stem like-cell and NG2+ progenitor-like cell subpopulations. Understanding why current treatment strategies only work in certain tumor cell subpopulations will allow intelligent design of chemotherapeutic strategies with anti-tumor effects that are greater than those of conventional therapies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0058. CONDITIONAL INACTIVATION OF SMARCB1 IN P0 PERMISSIVE MOUSE CELLS RECAPITULATES THE TUMOR SPECTRUM OF BOTH HUMAN SMARCB1-RELATED SCHWANNOMATOSIS AND RHABDOID TUMOR PREDISPOSITION SYNDROMES

Jeremie Vitte 1, Fabrice Chareyre 1, Anat Stemmer-Rachamimov 2, Marco Giovannini 1

Abstract

INTRODUCTION: Schwannomatosis is characterized by the onset of multiple schwannomas, without involvement of the vestibular nerve, which is diagnostic of neurofibromatosis type 2. Genetic analysis permitted the localization of mutations in the tumor suppressor gene SMARCB1/Snf5/Ini1 in patients with familial or sporadic schwannomatosis. Specific inactivating mutations of the SMARCB1/Snf5/Ini1 gene are also the hallmark of malignant rhabdoid tumors (MRT), aggressive pediatric cancers, and also lead to a familial cancer predisposition syndrome. The mechanisms by which SMARCB1/Ini1/Snf5 germline mutations predispose to schwannomatosis versus MRT are still unknown. METHOD: Schwannomas are benign neoplasms of the peripheral nerve sheath and are believed to have their origin in embryonic neural crest cells. We used conditional mutagenesis to investigate the role of Smarcb1 biallelic inactivation in mouse neural crest and Schwann cell lineage. In this model, Cre recombinase expression was under the control of the protein zero (P0) gene promoter. RESULTS: P0Cre;Smarcb1flox/flox mice showed reduced viability and at 5 months of age most of the mice presented tumors arising from different cranial nerves (olfactory, trigeminal, vestibulocochlear). Analysis of the tumor spectrum showed a continuum from schwannoma to MPNST, to MRT presenting one or combinations of histological features characteristic of these different tumor types.Rhabdoid cells were found in 90% of tumors emanating from the fronto-nasal region. Histologically these tumors appeared malignant and poorly differentiated, without areas of low grade tumor or better differentiation. Molecular analysis of tumors showed derepression of cyclin D1 as found in mouse and human MRT. CONCLUSIONS: Altogether, our data not only establish a new model of Smarcb1-related tumorigenesis, but also provide important insight into the cell of origin of MRT.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0059. TARGETING THE HISTONE H3.3-K27M MUTATION FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMAS

Rintaro Hashizume 1, Lu Yu-Jen 1, Maxwell Tom 1, Yuichiro Ihara 1, Xi Huang 1, Todd Waldman 2, Sabine Mueller 1, Nalin Gupta 1, David James 1

Abstract

INTRODUCTION: Diffuse intrinsic pontine gliomas (DIPGs) carry a dismal prognosis despite the use of aggressive multi-modality treatment. No significant advances in the survival of DIPG patients have been made over the last few decades, and new therapeutic approaches are desperately needed. Recent results have identified a somatic mutation in the H3F3A gene, resulting in replacement of lysine 27 by methionine in its encoded histone H3.3 protein (H3.3-K27M) that occurs primarily in DIPGs. We hypothesize that the expression of this mutant protein in DIPG provides a unique therapeutic opportunity for treating this cancer. METHOD: H3.3-K27M mutation status was determined by direct sequencing of two pediatric DIPGs and one pediatric GBM tumor, using DNA from cell lines established from surgical biopsies. Histone H3.3 lysine 27 (H3K27) methylation status was evaluated by western blotting with antibodies specific for mono-, di-, and trimethylated H3K27. Cell proliferation assays were performed on these cell lines, and additional glioma cell lines lacking the H3.3 mutation, in order to measure the response to pharmacological inhibition with GSK-J4, a selective inhibitor of H3K27 demethylase JMJD3. GSK-J4 inhibits JMJD3-induced H3K27 demethylation, resulting in an increase of H3K27 methylation. RESULTS: The H3.3-K27M mutation was identified in the two DIPG cell lines, but not in the pediatric GBM cells, nor in other adult glioma cells. H3.3-K27M mutant DIPG cells showed rapid growth in vitro, with a doubling time of approximately 30 hours. In contrast, a pediatric GBM cell line with wild-type H3.3 grew much more slowly, with a doubling time of 72 hours. H3.3-K27M mutant DIPG cells showed elevated H3K27 methylation in comparison to H3.3 wild-type glioma cells. GSK-J4 induced a marked dose-dependent inhibition of growth in H3.3-K27M mutant DIPG cells while H3.3 wild-type glioma cells showed no response to GSK-J4. CONCLUSIONS: Our findings support altered histone H3.3 K27 methylation as a result of the H3.3-K27M mutation, which may be associated with rapid tumor cell growth in vitro, and is potentially associated with selective tumor cell sensitivity to GSK-J4.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0060. SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONS) CONJUGATED WITH HEAT SHOCK PROTEIN Hsp70 FOR TARGETED DELIVERY TO MALIGNANT BRAIN TUMORS

Maxim Shevtsov 1, Ludmila Yakovleva 2, Boris Nikolaev 2, Anatoliy Dobrodumov 3, Kiril Onokhin 1, Natalia Bychkova 4, Anastasia Mikhrina 5, William Khachatryan 6, Irina Guzhova 1, Marina Martynova 1, Olga Bystrova 1, Alexander Ischenko 2, Boris Margulis 1

Abstract

INTRODUCTION: The superparamagnetic iron oxide nanoparticles (SPIONs) prepared by co-precipitation from Fe+2, Fe+3 salt solutions have the ability to function as theranostic agents. SPIONs act as powerful contrast agents for MRI due to their high magnetic moment, and both can be used as delivery vehicles for anti-cancer agents and thermotherapy. For increasing the therapeutic activity of SPIONs we conjugated the latter with heat shock protein Hsp70 that is well known for its immunomodulatory anti-tumor activity. The possibility for the targeted delivery of the conjugate was analyzed in series of in vitro and in vivo experiments. METHOD: Synthesized magnetic Hsp70 conjugates were analyzed by spectrophotometry, ELISA assays. Proton magnetic relaxation times T2 were measured with the help of the NMR-spectrometer (CXP-300, Bruker) in magnetic field 7.1 T. The in vitro binding and uptake of SPIONs and its Hsp70 conjugates was assessed on the C6 glioma cell culture by confocal and electron microscopy, flow cytometry assay. The in vivo traffic was analyzed in the model of intracranial C6 glioma. MR images (gradient echo (FLASH), T1- and T2-weighted, multi-sc and multi-echo (MSME T2-map) of rat glioma were obtained by Bruker Avance II NMR spectrometer 11 T. RESULTS: The parameters of SPIONs measured relaxivity corresponded to properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. According to the data from in vitro studies SPIONs were incorporated into C6 cells mostly by endocytosis pathway. Intriguingly, the conjugation of protein Hsp70 to the SPIONs increased the internalization of nanoparticles as was demonstrated by confocal microscopy and flow cytometry assay. In vivo studies confirmed the targeting ability of Hsp70 conjugates uptake and tumor contrast enhancement in comparison to non-coated nanoparticles. CONCLUSIONS: Magnetic nanoparticles based on the iron oxide represent a promising nanomaterial for the targeted therapy and imaging of malignant brain tumors. Covalent attachment of heat shock protein Hsp70 to the magnetic nanoparticles increased the uptake of SPIONs by C6 glioma cells and, what is more important, – provided the selectivity of tumor targeting in in vivo conditions. Thus, conjugation of SPIONs with the chaperone Hsp70 represents the attractive approach in the targeted therapy of malignant brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0061. MYC AMPLIFICATION STATUS INFLUENCES TUMOR IMMUNE EVASION IN MEDULLOBLASTOMA

Allison Martin 1, Christopher Nirschl 1, Magda Polanczyk 1, Kenneth Cohen 1, Drew Pardoll 1, Charles Drake 1, Michael Lim 1

Abstract

INTRODUCTION: Improvement in therapy for relapsed and refractory medulloblastoma represents an urgent unmet clinical need. Most tumors with this phenotype are in Group C, driven by MYC amplification. MYC amplification leads to a state of deregulated growth and increased metabolism. It is associated with an aggressive/invasive phenotype in medulloblastoma and other human cancers. PD-L1 is an immune regulatory molecule expressed on the cell surface of some solid tumors. Our group and others have shown that PD-L1 expression correlates strongly with outcome. However the interaction of MYC with immune regulatory molecules like PD-L1 has not been previously explored. METHOD: The expression of human PD-L1 and PD-L2 was evaluated via flow cytometry in human medulloblastoma cell lines with low MYC amplification, DAOY and UW228, and those with high MYC amplification, D283 and D425. Expression patterns of these molecules were recorded both at rest and after stimulation with 100units/ml of recombinant human interferon gamma. The same low MYC lines, DAOY and UW228, were transfected with a MYC overexpression plasmid and treated the same way. Finally DAOY was sorted using a FACS Aria into two populations based on PD-L1 expression, high versus low, and implanted into the flank of nude mice. RESULTS: This study demonstrates for the first time that human medulloblastoma cell lines express the immune regulatory ligands of PD-1, PD-L1 and PD-L2, both at baseline and in response to cytokine stimulation. The pattern of ligand expression correlates strongly with MYC amplification status. Tumors with constitutive ligand expression are generally negative for MYC amplification while those with inducible ligand expression are positive for MYC amplification. Artificially over-expressing MYC blunts the expression of both PD-L1 and PD-L2, changing the immune phenotype of the cell line. Tumors selected for high PD-L1 expression exhibited a growth disadvantage when compared to low expressers in vivo. CONCLUSIONS: These findings indicate that medulloblastoma has a unique immune phenotype corresponding with MYC expression patterns, and that MYC amplification may directly influence this tumor's ability to evade the host immune system. Expression of PD-L1 may confer a growth disadvantage, suggesting a novel role for the ligands of PD-1 in tumor growth and metabolism and lending a possible explanation for their decreased expression in the presence of MYC. Understanding the interaction between MYC and the host immune system could lead to additional therapies for MYC driven medulloblastoma and may have broader implications for the general role of MYC amplification in cancer.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0062. SENSITIVITY OF MEDULLOBLASTOMA TO RADIATION REQUIRES AN INTACT MITOCHONDRIAL APOPTOTIC PATHWAY

Andrew Crowther 1, Sha Chang 1, Hong Yuan 1, Mohanish Deshmukh 1, Timothy Gershon 1

Abstract

INTRODUCTION: Since the first successful use of radiation therapy (RT) for medulloblastoma in the 1950's, RT has been integral to medulloblastoma treatment. The molecular mechanisms that determine the sensitivity of medulloblastoma to RT remain unknown. Identifying why medulloblastomas typically respond to RT may yield critical insight into how to enhance tumor response and to predict treatment failure. METHOD: We have previously demonstrated that medulloblastomas have an inherent tendency to undergo programmed cell death that depends on the pro-apoptotic Bcl-2 family protein Bax. To test the role of Bax-dependent apoptosis in medulloblastoma response to RT, we administered 10Gy external beam radiation focused on the posterior fossa of SmoM2 mice with primary medulloblastoma with and without simultaneous deletion of Bax. We then examined the histologic response to treatment and the effect of treatment on animal survival. RESULTS: All Math-1 cre;SmoM2 mice developed medulloblastoma by 12 days of age and tumors in Bax-deficient Smom2 mice were equally aggressive. Medulloblastomas in Math-1 cre;SmoM2 mice were markedly sensitive to RT. Within 4 hours of treatment, almost all tumor cells demonstrated activated caspase 3 and by 5 days after treatment tumors had largely regressed. 40% of mice were long-term survivors. In contrast, Bax-deficient SmoM2 medulloblastomas were thoroughly radiation-resistant. Rather than undergoing apoptosis en masse, Bax-deficient tumor resolved within days into 2 distinct populations, as cells either differentiated as neurons or continued to proliferate. No SmoM2 mice with Bax deletion survived long-term. CONCLUSIONS: Our data highlight the absolute importance of intact apoptotic pathways to medulloblastoma treatment response. We show that the radiation sensitivity of medulloblastoma is not simply an effect of high proliferation rate. Importantly, we did not detect cell death through Bax-independent mechanisms such as mitotic collapse. Inability to initiate cell death programs may identify human patients who will not respond to standard therapy, while enhancing the tendency of cytotoxic therapies to trigger Bax-dependent cell death may increase therapeutic efficacy.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0063. TARGETING THE TRANSLATIONAL APPARATUS IN MYCN-DRIVEN MEDULLOBLASTOMA

Justin G Meyerowitz 1, W Clay Gustafson 1, Erin A Nekritz 1, Fredrik Swartling 2, Kevan M Shokat 1, Davide Ruggero 1, William A Weiss 1

Abstract

INTRODUCTION: Amplification of MYCN is associated with high-risk SHH-driven and group 4 medulloblastoma, while mis-expression and amplification of MYC is associated with aggressive group 3 tumors. The biology of these tumors, particularly the group 3/4 tumors, is poorly understood, with no effective targeted therapies. Published data by others shows that translational control downstream of the mammalian target of rapamycin (mTOR) is required for MYC-driven oncogenesis. mTOR signals through two primary outputs, rpS6 kinase (S6K) and the translation initiation factor eIF4E. A new class of clinical mTOR ATP-competitive inhibitors disrupts signaling through both effectors, whereas the allosteric binder rapamycin disrupts only S6K. METHOD: We have developed a model of group 4 medulloblastoma in which high levels of mutationally-stabilized NMYC protein drive oncogenesis. Transplantation of NMYCT58A-transduced cerebellar neurospheres into nude mice generates tumors with high penetrance. Here, we incorporate into this system mice with phosphorylation-deficient alleles of rpS6 or 4E-BP, the latter of which is inducible and disrupts downstream eIF4E activity. Using these strains, we will clarify the individual importance of signaling through S6K and 4E-BP in NMYCT58A-driven tumor formation. Furthermore, we use both cell-based assays and mouse models to determine whether MYC- and MYCN-expressing, SHH-independent medulloblastoma is sensitive to mTOR kinase inhibition. RESULTS: Transduction of NMYCT58A, but not wild-type NMYC or GFP control, into neurospheres derived from cerebellum, forebrain, or brainstem generated tumors when orthotopically transplanted into nude mice, and those generated from postnatal (P0) cerebellar neurospheres are SHH-independent. Transduction of NMYCT58A increases proliferation of P0 cerebellar neurospheres compared to transduction of either NMYCWT or GFP. Immunoblotting of NMYCT58A-transduced P0 neurospheres verifies that rapamycin decreases phosphorylation of rpS6, whereas the mTOR active site inhibitors PP242 and INK128 disrupt phosphorylation of both rpS6 and 4E-BP. Measurement of cell number shows that PP242 but not rapamycin inhibits proliferation of NMYCT58A-transduced neurospheres. CONCLUSIONS: The mechanistically distinct activities of mTOR allosteric vs. active site inhibitors have enormous therapeutic implications, as in our medulloblastoma model, active site inhibitors of mTOR, but not rapamycin, demonstrate efficacy. Taken together, this supports the idea that signaling through S6K is dispensable, whereas eIF4E is required for MYC-driven oncogenesis.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0064. GENE EXPRESSION ANALYSIS OF 163 PEDIATRIC LOW-GRADE GLIOMA ACROSS DIFFERENT AGES, LOCATIONS, HISTOLOGICAL SUBTYPES AND BRAF GENOMIC STATUS REVEALS ASSOCIATED MOLECULAR CHARACTERISTICS

Guillaume Bergthold 1, Benjamin Rich 1, Pratiti Bandopadhayay 1, Jennifer Chan 2, Sandro Santaga 2, Yujin Hoshida 3, Todd Golub 3, Barbara Tabak 3, Ruben Ferrer-Luna 1, Jacques Grill 4, Patrick Yung Wen 2, Charles Stiles 1, Mark Kieran 1, Keith Ligon 1, Rameen Beroukhim 1

Abstract

INTRODUCTION: Low-grade gliomas (LGGs), the most common pediatric brain tumors, constitute a broad and heterogenous group of diseases. Histologic diagnosis is often controversial due to the lack of reproductibility and the absence of correlation to clinical outcome. Although the recent identification of recurrent BRAF duplications and V600E point mutations in subsets of pediatric LGGs has brought new insights into genesis of these tumors, little is known about their molecular characteristics. The aim of this study was to describe the expression patterns of a large cohort of pediatric LGGs and to compare these patterns to patterns of expression in normal brains. METHOD: We performed gene expression profiling on 163 paraffin-embedded pediatric and 20 adult LGGs across 6100 selected genes known to be dysregulated in cancer. Unsupervised and supervised clustering methods were applied to distinguish specific molecular features associated with age, location, histologic subtype and BRAF genomic status of the tumors. We also performed multivariate analyses to specify differences associated with each clinical feature, and compared those differences with a large cohort of normal brain samples matched for location and age (Brainspan database). RESULTS: NMF (Non-negative Matrix Factorization) clustering on the 163 pediatric and 20 adult LGGs distinguished three molecular groups, associated with location, age, BRAF genomic status, and to a lesser extent histologic subtype. Differences between tumors with these clinical features were also showed using principal component analysis. Pediatric BRAF-duplicated tumors did not exhibit a distinct molecular pattern compared to BRAF wt tumors. After controlling for tumor location, we found that 75% of the genes differentially expressed between adolescent and adult LGGs were similarly differentially expressed between normal adolescent and adult brains (p< 0.0001). CONCLUSIONS: We report the largest cohort of expression profiles of pediatric LGGs and identify differences associated with age, location, histologic subtype and BRAF genomic status. Most of the molecular differences between adolescent and adult LGGs are reflected in normal brain development, suggesting that normal brain development from adolescence to adulthood may influence tumor behavior.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0065. EXPRESSION OF miR-203 IS ASSOCIATED WITH AGRESSIVE AN PHENOTYPE IN PEDIATRIC MEDULLOBLASTOMA

Rishi R Lulla 1, Joseph Laskowski 1, Monica Gireud 2, Jason Fangusaro 1, Stewart Goldman 1, Vidya Gopalakrishnan 2

Abstract

INTRODUCTION: MicroRNAs (miRNAs) are small non-coding RNAs that play various roles in biological processes including proliferation, differentiation and apoptosis. Altered expression of miRNAs contributes to the development and maintenance of medulloblastoma. Additionally, some miRNAs are over-expressed in metastatic medulloblastoma. In both prostate and breast cancer, miR-203 has been shown to be responsible for cell invasiveness. From our own previous experience, expression of miR-203 in pediatric ependymoma was identified as predictor of early recurrence. Therefore, we investigated the association of miR-203 expression with clinical outcomes in a pediatric medulloblastoma. METHOD: Primary tumors and corresponding clinico-pathologic data were collected on 48 patients with medulloblastoma. RNA purification of tumors was done with RecoverAll™ RNA isolation kit. The purified total RNA was used for cDNA synthesis with primers for miR-203 and mamm-u6 as a control. TaqMan® miRNA assays were done in triplicate to quantify the levels of each target using the Applied Biosystems 7900HT Fast Real-Time PCR system in 96-well plates. Cycle threshold (Ct) values under 35 were considered positive for the presence of target miRNAs. Expression of miR-203 across samples was compared using delta-Ct. ExpressionSuite® software was used for data analysis. RESULTS: Levels of miR-203 varied considerably in our cohort, with a median delta-Ct of 10.87. Among tumors with large cell/anaplastic histology (n = 12), 9 had a high relative expression of miR-203 with a delta-Ct value below the median. Similarly, high miR-203 levels were observed in 8 of 11 patients with metastatic disease, regardless of histologic subtype. No correlation between miR-203 expression levels and patient age or overall survival was observed. Medulloblastoma subgrouping for this cohort is underway as is determination of miR-203 expression of normal cerebellum for comparison and will be presented. CONCLUSIONS: Our preliminary results suggest that miR-203 is associated with an aggressive phenotype in medulloblastoma. Tumors with large cell/anaplastic histology and those with invasion into other central nervous structures had the highest expression of miR-203 in our cohort. Future studies include: identification and validation of downstream targets of miR-203 as well as determination of medulloblastoma subgroup specific miR-203 expression levels. We hypothesize that miR-203 may be implicated in invasion and migration in medulloblastoma as it is in other tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0066. IDENTIFICATION OF MOLECULARLY AND CLINICALLY DISTINCT SUBGROUPS OF CHOROID PLEXUS CARCINOMAS

Diana Merino 1, Adam Shlien 1, Margaret Pienkowska 1, Uri Tabori 1, Richard Gilbertson 2, David Malkin 1

Abstract

INTRODUCTION: Choroid plexus tumors are a group of rare intraventricular tumors arising in young children. While choroid plexus papillomas (CPPs) and atypical choroid plexus papillomas (aCPP) exhibit favourable outcomes, choroid plexus carcinomas (CPCs) exhibit variable outcomes; tumor progression and relapse is observed in ∼60% of patients, and most survivors experience significant long-term developmental and cognitive deficits. We studied a large cohort of choroid plexus tumors using an integrative high-throughput molecular approach to characterize the molecular profile and refine the current classification of these poorly understood tumors. METHOD: We hybridized 55 choroid plexus tumors with known TP53 status to Affymetrix Human Exon 1.0 ST expression, and SNP 6.0 genotyping microarrays. We analyzed gene expression profiles and copy number aberrations, and distinguished between molecularly distinct tumor subclasses. Molecular and clinical data were correlated to uncover relationships between newly defined subgroups and clinical outcomes. RESULTS: Copy number alterations expanded entire chromosomes, suggesting the prevalence of aneuploidy in most tumor samples. Chromosome-wide copy number and gene expression profiles segregated CPCs from the benign CPPs and aCPPs. The molecular profile of CPCs was further refined into subgroups harboring a mutant versus a wildtype TP53. In mutant TP53 CPCs, chromosome-wide copy number profiles distinguished between two subgroups with significantly distinct clinical outcomes. Hyperdiploid mutant TP53 CPCs exhibited a less favourable long-term overall survival compared to the hypodiploid subgroup (3-year OS: 0% vs. 83% in hypodiploid mutant TP53 CPCs, p = 0.005). CONCLUSIONS: Our integrative study has characterized the recurrent chromosome-wide alterations and gene expression profiles found in choroid plexus tumors, and further identified novel molecular subgroups in CPCs with significantly distinct survival outcomes. Our findings suggest that CPCs can be further classified into clinically-relevant subgroups to improve the clinical management of these malignant tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0067. TARGETING Wee1 FOR THE TREATMENT OF PEDIATRIC HIGH-GRADE GLIOMAS

Sabine Mueller 1, Rintaro Hashizume 1, Xiaodong Yang 1, Ilan Kolkowitz 1, Aleksandra Olow 1, Joanna Phillips 1, Ivan Smirnov 1, Maxwell Tom 1, Michael Prados 1, Mitchel Berger 1, Nalin Gupta 1, Daphne Haas-Kogan 1

Abstract

INTRODUCTION: Wee1 is a key cell cycle regulatory kinase. In this study we evaluated Wee1 expression in pediatric gliomas to assess its relevance as a therapeutic target when used in combination with radiation therapy. Based on our expression results, which showed increasing Wee1 expression in association with increasing glioma malignancy grade, we investigated the specific Wee1 inhibitor MK-1775 for in vitro and in vivo efficacy when administered concurrently with radiation for the treatment of pediatric high-grade gliomas (HGG), including diffuse intrinsic pontine gliomas (DIPG). METHOD: Gene expression analysis was performed for 38 primary pediatric gliomas (3 grade 1, 10 grade 2, 11 grade 3, 14 grade 4) and 8 normal brain samples using the Agilent 4x44K array. Clonogenic survival assays were carried out in HGG cell lines (n = 6). DNA repair capacity was evaluated by measuring levels of γ-H2AX, a marker of double strand DNA breaks. In vivo activity of MK-1775 with radiation was assessed in two distinct orthotopic engraftment models of pediatric HHG: one derived from a genetically engineered mouse carrying BRAFV600E mutation, and the other a xenograft model utilizing cells from a patient's DIPG. RESULTS: Wee1 is overexpressed in pediatric HGGs, especially in DIPG, with positive correlation between increasing Wee1 expression and higher malignancy grade (p = 0.007 for grade 3 + 4 vs. 1 + 2). Combination treatment with MK-1775 and radiation, in vitro, reduced clonogenicity and increased expression of g-H2AX in HGG cells to a greater extent than achieved by radiation alone. Finally, combined MK-1775 and radiation conferred greater survival benefit to mice bearing engrafted, orthotopic HGG and DIPG tumors, than was achieved by treatment with radiation alone (BRAFV600E allograft model p = 0.0061 and DIPG brainstem xenograft model p = 0.0163). CONCLUSIONS: Our results highlight MK-1775 as a promising new therapeutic agent for use in combination with radiation for the treatment of pediatric HGGs, including DIPG. To our knowledge this is the first investigation that reports on the expression of Wee1 in all grades of pediatric glioma, including DIPG, and uses relevant pediatric glioma models to assess the effect of MK-1775 in combination to radiation in vivo. Our results have motivated a soon-to-be-initiated phase 1 clinical trial for newly diagnosed DIPG.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0068. FLUORESCENCE-GUIDED SURGERY WITH 5-AMINOLEVULINIC ACID FOR RESECTION OF BRAIN TUMORS IN CHILDREN

Thomas Beez 1, Sevgi Sarikaya-Seiwert 1, Gisela Janssen 2, Jörg Felsberg 3, Hans-Jakob Steiger 1, Daniel Hänggi 1

Abstract

INTRODUCTION: After exogenous administration, 5-aminolevulinic acid (5-ALA) is metabolized in tumor cells, where its major metabolite accumulates. Irradiation with light of a specific wavelength induces fluorescence. This mechanism can be exploited to differentiate between normal and neoplastic tissue during surgery. A RCT established the impact of 5-ALA on gross total resection and prognosis in adult glioblastoma. As the impact of radical resection on outcome of pediatric brain tumors is evident, 5-ALA could be beneficial. Two case reports of fluorescence-guided surgery in children were published. We report our experience with 5-ALA in children harboring WHO grade I, III and IV brain tumors. METHOD: 10 patients with a mean age of 11 years (range 4-15 years) harboring brain tumors with infiltrative appearance on preoperative MRI received 5-ALA according to the previously published adult protocol. Informed consent of parents was obtained on the basis of individual treatment attempts. Patients orally received 20mg/kg bodyweight 5-ALA 2-4 hours before induction of anaesthesia. During surgery, the neurosurgeon repeatedly switched the microscope (OPMI Pentero, Carl Zeiss, Germany) into fluorescence mode to inspect the resection cavity for positive fluorescence. Routine blood samples were taken and clinical monitoring for adverse events was performed. The local ethics committee approved this retrospective analysis. RESULTS: Histology revealed pilocytic astrocytoma WHO grade I (n = 6), classical medulloblastoma WHO grade IV (n = 3) and anaplastic ependymoma WHO grade III (n = 1). Positive fluorescence of tumor tissue was observed in none of these cases. Significant increases were registered for alanine aminotransferase (14.9 U/l vs. 37.7 U/l, p < 0.0001) and gamma glutamyl transpeptidase (12.7 U/l vs. 39.3 U/l, p < 0.0001). Renal and hematological parameters remained stable. No adverse reactions were evident on clinical examination. CONCLUSIONS: Positive fluorescence was not observed in this pediatric series. Adverse reactions were similar to those reported for adults. The value of fluorescence-guided surgery appears ambiguous in the treatment of pediatric brain tumors. While negative results in pediatric WHO grade I tumors correspond to observations in adult low-grade glioma, the lack of fluorescence in WHO grade III and IV tumors remains unexplained. Apart from tumor-specific aspects, pharmacokinetic differences in children might account for negative findings. Further studies are required to elucidate pharmacokinetic and pharmacodynamic particularities of 5-ALA in children and to assess its role in the resection of pediatric brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0069. EXPERIMENTAL COMBINATION THERAPY: HISTONE DEACETYLASE AND DNA METHYLTRANSFERASE INHIBITORS IN BRAIN CANCER CELL MODEL

Ana-Maria Marino 1, Ninib Baryawno 1, John-Inge Johnsen 1, Arne Ostman 1

Abstract

INTRODUCTION: Gene expression is controlled by several epigenetic mechanisms; one involves DNA methylation which is regulated by distinct but related methyltransferases. Another mechanism involves the acetylation and deacetylation of histone and non-histone proteins that control the transcription and regulation of genes involved in proliferation, cell cycle control, survival, DNA repair and differentiation. Investigation shows that aberrant epigenetic alterations can be reverted with the use of epigenetic modificators. Several clinical studies of combination therapy in hematological malignancies have improve the response rates of patients. Therefore the use of DNMTs inhibitors and HDAC inhibitors for the treatment of solid tumors seems like a very promising strategy. METHOD: We have combined DNMT inhibitor 5-Aza-2′deoxycytidine and sodium 4-Phenylbutyrate using medulloblastoma cell models, and have noticed that the combination with imatinib and sorafenib results in significant cell killing at very low dosages. We have checked for caspase 3, and 7 activity using a luciferase based assay. HDAC and DNMT activity has been checked using an anzyme based assay. Expression of HDAC and DNMT by western blotting. DNA methylation before and after treatment using an enzyme system. Acetylation by Elisa and Western Blotting. RESULTS: We observed an increased activity of caspases 3, and 7 after triple treatment, and a decrease in cell viability. The activity levels of HDACs differs according to to therapy. The activity of DNA methyltransferases also changes and we are currently checking on the expression of DNMT1, DNMT3A, and DNMT3B. We have checked for changes in DNA global methylation and observed a change towards hypomethylation and interesting observation was that imatinib alone gave a change in methylation. Currently we are checking for changes in acetylation after triple combination. We are also currently conducting some in vitro radiation studies after triple treatment. CONCLUSIONS: Our results suggest that these drugs act together to enhanace cell killing most likely by appoptosis. Although this was an in vitro study our results duggest that by reverting methylation and acetylation we are enhancing cell death and overcoming resistance to target therapy. We suggest that these drug combination and therefore should be further tested for their precise molecular activivity in preclinical models of medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0070. PROTEOGLYCANS IN PEDIATRIC ASTROCYTOMA

Anna Wade 1, Jane R Engler 1, Aaron E Robinson 1, Joanna J Phillips 1

Abstract

INTRODUCTION: Proteoglycans, components of the cell microenvironment, regulate cell signaling and migration by interacting with growth factor receptors, extracellular ligands, the extracellular matrix, and intracellular structural proteins. In diverse cancers, including brain cancer, proteoglycans have been shown to regulate multiple oncogenic pathways and promote tumor growth, invasion, inflammation and angiogenesis. As proteoglycans and their enzymatic modification may be important in disease, and may represent novel therapeutic targets and tumor biomarkers, we examine the expression of proteoglycan core proteins and their related biosynthetic and modifying enzymes in pediatric astrocytoma. METHOD: Gene expression data was obtained from a total of 26 astrocytomas at UCSF as described previously using Agilent 4x44 arrays (G42514F): pilocytic astrocytoma (n = 6), anaplastic astrocytoma (AA) (n = 8) and glioblastoma (GBM) (n = 12) (Engler J. et al, 2012). Non-neoplastic brain samples resected for epilepsy were derived from pediatric (n = 2) and adult (n = 6) patients. Expression data is displayed as mean log2(tumor/normal) and compared to data in adult GBM (Wade A. et al., 2013). Genes of interest were analyzed using unpaired Mann Whitney U test or Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. RESULTS: Pilocytic astrocytomas have a marked increase in expression of the proteoglycan core protein genes ACAN and PTPRZ1 (ANOVA p = 0.0028, 0.0004 respectively). In contrast, in high-grade astrocytomas, AA and GBM, the gene expression of CD44 was significantly increased and in GBM the expression of GPC5 was significantly decreased (ANOVA p = 0.003, 0.008 respectively). A comparison of gene expression alterations between pediatric pilocytic astrocytoma, pediatric high-grade astrocytoma, and adult GBM, reveals multiple similarities including increased expression of the extracellular heparan sulfate (HS) endosulfatase genes SULF1 and SULF2, which act to change the sulfation status and binding properties of HS. CONCLUSIONS: Pediatric astrocytomas exhibit multiple alterations in the expression of proteoglycan core proteins and proteoglycan synthetic, modifying and degrading enzymes. In some cases, the alterations in pediatric tumors mirrored alterations previously identified in adult GBM, such as increased expression of the extracellular endosulfatases, increased expression of the 3S HS sulfotransferases, and decreased expression of the 6S HS sulfotransferases, suggesting potential roles for HS sulfation in disease. Proteoglycans and their modifying enzymes represent potential extracellular therapeutic targets and tumor biomarkers in pediatric glioma. Ongoing studies in the laboratory are investigating these possibilities and examining proteoglycan expression at the protein level.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0071. IDENTIFICATION OF TRANSCRIPTOMIC AND EPIGENETIC SUBGROUPS OF SUPRATENTORIAL EPENDYMOMAS

Hendrik Witt 1, Martin Sill 3, Stephen C Mack 4, Khalida M Wani 5, Sally Lambert 6, Theophilos Tzaridis 1, Sebastian Bender 1, David TW Jones 1, Till Milde 7, Paul A Northcott 1, Marcel Kool 1, Andreas von Deimling 8, Andreas E Kulozik 2, Olaf Witt 7, Peter Lichter 10, V Peter Collins 6, Ken Aldape 5, Michael D Taylor 4, Andrey Korshunov 9, Stefan M Pfister 1

Abstract

INTRODUCTION: Recently, ependymomas were classified into molecular subgroups based on transcriptomic alterations. Focussing on tumors localized within the posterior fossa, two distinct entities of ependymoma were delineated by several studies, which also show striking differences in genetic characteristics and clinical outcome. So far, subgrouping approaches for supratentorial ependymomas have been lacking clinical and prognostic data, and have been limited to small cohorts. To develop an ependymoma consensus of clinically relevant molecular subgroups analogous to medulloblastoma, a broadly accepted molecular classification scheme is desperately needed. METHOD: We studied genome-wide mRNA expression profiling (Agilent, 44k) and DNA methylation changes (Illumina, 450k) in 65 primary supratentorial ependymomas. When performing unsupervised clustering, we identified three major clusters, which displayed significant overlap when comparing transcriptome-based and methylation-based subgrouping. We integrated these findings with previously detected cytogenetic aberrations. Three independent expression profiling data sets from previously published ependymoma studies (Johnson et al.; Wani et al.; Witt et al.) were being used as validation cohorts. For validation of single molecular markers, an independent set of 120 primary supratentorial ependymomas was available on tissue microarrays. RESULTS: Supratentorial ependymomas can be classified into three principle molecular subgroups, by whole transcriptome or whole methylome profiles. Subgroup 1 consists of adolescent patients and young adults. None of these patients died, and high-risk aberrations such as gain of chromosome 1q and deletions of CDKN2A were absent. Patients belonging to Subgroups 2 and 3 have a much worse outcome, and comprise predominantly children and infants. These tumors often show stable genomes, with some harboring high-risk cytogenetic aberrations including gain of chromosome 1q and deletion of CDKN2A. CONCLUSIONS: In summary, we could decipher molecular subgroups of supratentorial ependymoma using genome-wide trancriptome and methylome approaches. These subgroups show a close overlap with recently published transcriptome-based analyses across all locations. Our results suggest that robust molecular subgroups of ependymoma with very distinct demographics and clinical course can be readily distinguished, and should therefore be considered in a consensus molecular classification of ependymoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0072. USP37 IS A NOVEL DEUBIQUITYLASE WITH TUMOR SUPPRESSIVE FUNCTION IN MEDULLOBLASTOMA

RJ Hatcher 1, CM Das 1, V Datar 1, P Taylor 1, A Singh 1, D Lee 1, G Fuller 1, L Ji 1, J Fangusaro 2, V Rajaram 2, S Goldman 2, C Eberhart 3, V Gopalakrishnan 1

Abstract

INTRODUCTION: Expression of the RE1-Silencing Transcription Factor (REST), a repressor of neurogenesis, is elevated in the pediatric brain tumor, medulloblastoma. Elevated REST expression is associated with diminished tumor differentiation and poor overall/event-free survival in patients. REST loss prevents tumor formation in mouse xenograft models, whereas its constitutive expression in neural progenitors contributes to tumor development. Here, we demonstrate a novel role for REST in the control of cell proliferation. We have identified an unexpected link between REST and a deubiquitylase called USP37, in the proteasome-dependent destabilization of the cyclin-dependent-kinase-inhibitor (CDKI) p27 and deregulation of cell proliferation and medulloblastoma development. METHOD: REST knockdown and constitutive USP37 expression was achieved by transient transfection of human medulloblastoma cells and analyzed by qRT-PCR and Western blotting. REST binding to target genes was assessed by chromatin immunoprecipitation assays. Interaction between USP37 and p27 was measured by co-immunoprecipitation and in vitro deubiquitylation assays. USP37 dependent tumor suppression was measured using mouse orthotopic models and tumors analyzed by H&E staining and immunohistochemistry. Human tumor samples and mouse cerebella were stained and REST, USP37 and p27 levels determined by immunofluorescence assay. RESULTS: REST loss in human medulloblastoma cells and its constitutive expression in neural progenitors decreased and increased cell proliferation respectively. REST knockdown increased p27 protein levels and concomitantly derepressed USP37 transcription through loss of REST binding to the cognate promoter. Constitutive wild-type (WT) USP37 but not mutant USP37 expression rescued REST-associated p27 destabilization, blocked cell proliferation and promoted neurogenesis. Constitutive expression of WT USP37 in tumor cells also decreased their tumorigenic potential in the murine cerebellum compared to vector or mutant USP37 expressing tumor cells. Significantly, genetic and pharmacological targeting of REST-associated epigenetic activity similarly upregulated USP37 expression and blocked tumorigenesis. CONCLUSIONS: Our work has identified a novel role for the repressor of neurogenesis REST, in the control of medulloblastoma cell proliferation. We have shown that REST represses the transcription of a previously unknown p27-specific deubiquitylase called USP37, which results in p27 destabilization and sustained tumor cell proliferation in vitro and tumorigenesis in vivo. Genetic or pharmacological upregulation of USP37 blocks tumor growth suggesting that USP37 is a novel medulloblastoma tumor suppressor. Consistent with this, human tumor samples exhibit reciprocal of REST and USP37/p27. Thus, we have identified USP37 as a novel tumor suppressor and a potential target for medulloblastoma therapy.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0073. SUSCEPTIBILITY TO IMPAIRED ASYMMETRIC DIVISIONS LEADS TO HIGH-GRADE ASTROCYTOMA FORMATION IN RESPONSE TO BRafV600E EXPRESSION AND Ink4a/Arf DELETION

Amelie Griveau 1, Robin Lerner 1, Rebecca Ihrie 2, Sista Sugiarto 1, Yuichiro Ihara 1, Brian Reichholf 1, Emmanuelle Huillard 3, Martin Mcmahon 1, David James 1, Joanna Phillips 1, Arturo Alvarez Buylla 1, David Rowitch 1, Claudia Petritsch 1

Abstract

INTRODUCTION: Pediatric malignant astrocytomas (MA) are a leading cause of mortality for pediatric patients with central nervous system cancer. An activating mutation of BRAF (BRAFT1799A encoding BRafV600E) is present in 23% of pediatric diffuse astrocytomas (WHO grades II-IV; Cancer Res. 70:512-9; 2010). Importantly, concomitant homozygous deletion of CDKN2A, which encodes the INK4A and ARF tumor suppressors, defines a subset of BRafV600E high-grade pediatric glioma (Clin. Cancer Res. 17:7595-604; 2011). BRafV600E transforms Ink4a/Arf-deficient but not wild-type embryonic mouse neural progenitors in vitro and produces high-grade astrocytoma in vivo when these cells are orthotopically transplanted into SCID mice (PNAS. 109:8710-5; 2012). METHOD: To follow tumor development in situ, we injected adenovirus Cre in the lateral ventricle (LV) wall of 2-3 months mice to simultaneously activate BRafV600E and inactivate Ink4a/Arf. Adenoviral Cre expression was Gfap regulated to target type B cells. We analyzed the brains of injected mice by immunohistopathology at defined times: 4 and 21 days postinjection, and at tumor formation. This approach allowed us to target a restricted number of cells, and to spatially and temporally follow the process of transformation. RESULTS: We find that activation of BRafV600E alone in NSCs promotes gliogenesis at the expense of neurogenesis, whereas concomitant induction of BRafV600E and deletion of Ink4a/Arf causes formation of high-grade astrocytoma adjacent to the LV. At 21 days post-injection, oligodendrocyte precursor cells (OPCs) were highly proliferative. Olig2+ cells grew expansively, and under differentiation conditions proliferative NG2 + Olig2+ cells expanded at the expense of mature oligodendrocytes. The mutations disrupted asymmetric segregation and promoted symmetric distribution of NG2, EGFR and CD15 in vitro, arguing for early alteration of asymmetric OPC divisions. Injection of Olig2+ BRafV600E Ink4a/Arf-deficient cells led to orthotopic tumor formation. CONCLUSIONS: We propose that BRafV600E expression in Ink4a/Arf deficient Gfap+ cells boosts the expansion of OPCs, in part by deregulation of their asymmetric division, thereby impacting their differentiation into oligodendrocytes, and contributing to their astrocytoma transformation. Our data further suggest that NSC and early OPC are an equivalent origin of MA. Lastly, disrupted asymmetric divisions appear to be a general defect associated with progenitor cell transformation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0074. PLACENTAL GROWTH FACTOR/NEUROPILIN 1 SIGNALING IS A THERAPEUTIC TARGET IN PEDIATRIC MEDULLOBLASTOMA

Matija Snuderl 1, Ana Batista 2, Nathaniel Kirkpatrick 2, Carmen Ruiz de Almodovar 3, Lars Riedemann 2, Ellen Knevels 3, Thomas Schmidt 3, Teresa Peterson 2, Sylvie Roberge 2, Carlos Bais 4, Stephen Yip 5, Martin Hasselblatt 6, Claudia Rossig 6, Napoleone Ferrara 4, Michael Klagsbrun 7, Dan Duda 2, Dai Fukumura 2, Lei Xu 2, Peter Carmeliet 3, Rakesh Jain 2

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant pediatric brain tumor and constitutes 20% of all brain tumors in children. The standard of care includes initial surgery followed by radiation and chemotherapy. Current regimens have demonstrated good survival rates even in high-risk populations. However, the treatment entails devastating morbidity, including cerebral mutism syndrome, decline in cognition and intellect, secondary malignancies and infertility and growth problems. Therefore, there is an urgent need for new therapies in children with medulloblastoma that provide not only increased survival in non-responders but also reduce the significant morbidity associated with current treatments. METHOD: We analyzed 72 clinical samples of medulloblastoma for expression of Placental Growth Factor (PlGF) and its receptors Nrp1 and VEGFR1. Samples were classified based on histology and molecular criteria using the immunohistochemical panel (IHC), array comparative genomic hybridization, and deep gene sequencing. We used 2 orthotopic human medulloblastoma models, D283MED and D341MED, and transgenic spontaneous Smo+/+ medulloblastoma model and inhibited PlGF and Nrp1 using genetic inhibition by shRNA and species specific antibodies (aPlGF, aNrp1) in vivo. We followed tumor growth and survival, circulating biomarkers and analyzed underlying molecular changes at the end of the study. RESULTS: Approximately 90% of medulloblastomas expressed PlGF across all WHO subtypes and all main molecular subtypes of medulloblastoma. PlGF was produced by stromal cells in response to stimulation by tumor secreted Shh. Inhibition of PlGF/Nrp1 signaling axis led to apoptosis of medulloblastoma cells via MAPK pathway. aPlGF treated mice survived significantly longer both in the D283-MED (p < 0.0001) and D341-MED (p < 0.0001) models. aPlGF treated Smo+/+ mice had significantly smaller tumors and preserved body weight (p = 0.047 and p = 0.0044, respectively). aNrp1 therapy led to tumor regression and improved survival compared to controls (p < 0.01). CONCLUSIONS: PlGF and Nrp1 are required for the growth and spread of medulloblastoma. PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased spinal metastases, and increased mouse survival. Majority of PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and PlGF acts through Nrp1 signaling - but not vascular endothelial growth factor receptor 1 (VEGFR1)-to maintain tumor cell survival. Together with the reported minimal side effects of aPlGF and aNrp1 blockade from adult Phase I clinical trials, these findings identify PlGF and Nrp1 as potential targets for therapy of pediatric medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0075. THE MODULATION OF mTOR/HIF-1α PATHWAY IS EFFICIENTLY INHIBITING PEDIATRIC MALIGNANT BRAIN TUMOR CELL LINES

Aurelia Nguyen 1, Erwan Pencreach 1, Christelle Lasthaus 1, Viviane Lobstein 1, Eric Guerin 1, Dominique Guenot 1, Natacha Entz-Werle 2

Abstract

INTRODUCTION: Intratumoral hypoxia plays a fundamental role in tumor progression and resistance to therapies. Tumor cell adaptation to hypoxic environment is regulated partially by the Hypoxia Inducible Factor-1 (HIF-1), which is involved in gene expression of the cell energy metabolism, tumor angiogenesis or metastatic processes. HIF-1 accumulation in cells is driven in particular by mTor pathway activation. Recently, studies demonstrated that concomitant inhibition of HIF-1 plus mTOR is completely suppressing HIF-1 accumulation. Interestingly, these targets has been implicated in several pediatric brain tumors. Therefore, preclinical studies were launched to understand the efficacy of such inhibition in brain tumor cell lines. METHOD: For this study, 3 high-grade glioma (HGG) and 2 DIPG (diffuse intrapontine glioma) patient-derived cell lines were developed. They were cultured in normoxic and hypoxic conditions and, to go further, were xenografted subcutaneously in nude mice. For one HGG, the cell line was obtained before and after the combination (rapamycin + irinotecan) given to the patient. To test the impact of mTor/HIF-1 inhibition with irinotecan + rapamycin, in vitro assays were performed, analyzing the drug effects, alone or in combination, with clonogenic and proliferation evaluations. The xenografted tumors were also treated and, to understand the tumor sensitivity, protein and glycolytic analyses were done. RESULTS: The drug combination reduced cell proliferation in all cell lines except for the cell line, deriving from a pre-treated patient. This effect was only observed in hypoxic conditions and the combination abrogated completely HIF-1 expression. In vivo experiment was only done for 2 HGGs because of a slow tumor growth (more than 4 months), the tumors disappeared with the drug combination. Histological analyses showed a major tumor destruction, which was correlated to tumor anti-glycolytic and anti-angiogenic effects. The molecular screening of the cell line, deriving from the pre-treated patient, will help us to understand the molecular pathway modulations after treatment. CONCLUSIONS: Our results demonstrated the efficacy of this new targeted strategy in pediatric HGG. These promising results encouraged already the development of a phase I study in the pediatric setting including refractory tumors and especially pediatric brain tumor (half of the including patients).

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0076. MECHANISM OF ACTION AND THERAPEUTIC APPLICATION OF AURORA KINASE B INHIBITION IN c-MYC OVEREXPRESSING MEDULLOBLASTOMA

Roberto Diaz 1, Brian Golbourn 1, Claudia Faria 1, David Shih 1, Danielle MacKenzie 1, Daniel Picard 1, Melissa Bryant 1, Christian Smith 1, Michael Taylor 1, Annie Huang 1, James Rutka 1

Abstract

INTRODUCTION: Aurora kinase B regulates multiple steps in mitosis including the completion of cytokinesis. Aurora B inhibition triggers G2/M and post-mitotic checkpoints and has been demonstrated to elicit apoptosis in multiple cancer cell types. Since cells that overexpress c-MYC are uniquely sensitive to agents that trigger G2/M checkpoints, we hypothesized that medulloblastoma cells which overexpress c-MYC would be sensitive to the effects of Aurora B inhibition. METHOD: The MYC mRNA expression in sub-grouped medulloblastoma samples (n = 117) was compared to normal cerebellum. Specific inhibition of Aurora B with AZD1152-HQPA was determined by Western blotting. Cell counts, microscopy, FACS DNA content analysis, and Western blotting were used to assay the effects of Aurora B inhibition in c-MYC overexpressing medulloblastoma cells versus isogenic controls. The transcriptional response to Aurora B inhibition was assessed by mRNA microarray and analyzed using biological process gene ontology GSEA and gene enrichment mapping. D458 (MYC amplified medulloblastoma) cerebellar xenografts were monitored using bioluminescence imaging. Animals received AZD1152-HQPA (50 mg/kg/day) or vehicle subcutaneously for 21 days. RESULTS: MYC mRNA is highest in Wnt and Group 3 medulloblastoma when compared to adult or fetal cerebellum. Medulloblastoma cells overexpressing c-MYC undergo apoptosis after 48 hours of Aurora B inhibition and show downregulation of a DNA binding genes compared to isogenic control. Change in tumor photon flux from day 7 to day 14 was 3687 ± 913.3 SEM% in control versus 729.3 ± 93.68 SEM% with drug treatment (N = 11/group, P < 0.009). The mean survival time was 18 days (17 – 18 days, 95% CI) for control and 34 days (27 – 40 days, 95% CI) for AZD1152-HQPA treated animals (N = 5/group, P < 0.003). CONCLUSIONS: Aurora kinase B inhibition with AZD1152-HQPA triggers a unique transcriptional response in c-Myc overexpressing medulloblastoma cells. The growth of medulloblastoma endogeneously expressing high levels of c-Myc is impaired by Aurora B inhibition in-vivo resulting in prolonged survival. This suggests the potential for use of Aurora B inhibitors as subgroup specific chemotherapeutic agents in patients with Group 3 medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0077. ONCOLYTIC POLIOVIRUS IMMUNOTHERAPY OF PRIMARY CNS TUMORS

Matthias Gromeier 1, Annick Desjardins 1, John H Sampson 1, Stevie JE Threatt 1, James E Herndon 1, Allan Friedman 1, Henry S Friedman 1, Darell D Bigner 1

Abstract

INTRODUCTION: The poliovirus receptor Necl5/CD155, an onco-fetal cell adhesion molecule widely expressed in cancer, is universally associated with pediatric medulloblastomas/gliomas and adult glioblastomas. Therefore, primary explant tumor cells from patients are infected and rapidly killed by poliovirus. The main detriments to clinical use of poliovirus are its grim neuropathogenic profile and its inherent genetic instability. We resolved both obstacles through genetic recombination with human rhinovirus. The resulting recombinant, PVS-RIPO, is neuron-incompetent, due to translation restrictions in the normal CNS, but fully replication-capable in CNS tumor cells, due to constitutively active MAPK signals to translation machinery. METHOD: PVS-RIPO was cGMP manufactured by NCI, tested in extensive IND-directed toxicology studies in macaques, FDA approved (IND no. 14,735) and IRB approved; Phase I/II clinical trials in adult patients with recurrent glioblastoma were recently initiated at the Preston Robert Brain Tumor Center at Duke (NCT01491893). Administration is by convection-enhanced intracerebral/intratumoral infusion. Five patients have been treated on this protocol with rapid dose escalation to the max. feasible dose (10e10 TCID). In parallel, mechanistic studies of poliovirus oncolytic activity and the resulting host inflammatory response have been conducted in vitro and in syngenetic, immune-competent mouse models for glioma. RESULTS: Early indications from our clinical trial are that PVS-RIPO is exceedingly well tolerated in this patient population, without any evidence for encephalitis, meningitis or poliomyelitis. Importantly, there are promising signs of clinical improvement and radiological responses in multiple patients (>9 months post infusion). Mechanistic studies in vitro and in animal tumor models indicate that initial, direct viral tumor cell killing is critical, but that the host immunological response to tumor cell infection and -killing may constitute the main mechanism of clinical efficacy. Our early clinical results are consistent with these empirical findings. CONCLUSIONS: Our early clinical observations suggest that oncolytic virotherapy with PVS-RIPO may be promising for the treatment of primary CNS tumors in children and in adults. Major aspects of safety, efficacy and mechanisms of action, which were established in basic mechanistic studies in vitro, in animal tumor models, and in non-human primates, appear to be corroborated in our clinical studies. Of central importance for future endeavors is the identification of the immunologic response that results from PVS-RIPO mediated tumor infection and tumor cell killing in patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0078. IDENTIFICATION OF THE microRNAs CONTRIBUTING TO THE REGULATION AND MOLECULAR SPECIFICITIES OF THE MEDULLOBLASTOMA SUBGROUPS

Florence MG Cavalli 1, A Sorana Morrissy 1, Yisu Li 2, Andy Chu 2, Marc Remke 1, Nina Thiessen 2, Andrew J Mungall 2, Gary D Bader 3, David Malkin 1, Marco A Marra 2, Michael D Taylor 1; for the M.A.G.I.C Consortium

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant childhood brain tumor. Recently our understanding of the molecular biology of this disease has advanced dramatically with the identification of four molecular subgroups. These subgroups were identified based on distinct mRNA transcriptional profiles. However, the contribution of microRNAs (miRNAs) as regulators of these transcriptional landscapes remains unclear. Importantly, miRNAs may perturb the expression of multiple targets at once, and can act on specific tumor suppressors or oncogenes. As part of the MAGIC project, we are using next-generation sequencing approaches to comprehensively study the expression patterns of miRNAs in the different subgroups. METHOD: We performed miRNA-seq on 485 medulloblastoma primary tumors, and calculated the expression of every known miRNA present in miRBase. We clustered tumors using the Non-Negative Matrix Factorization (NMF) method, and identified differentially expressed miRNA and protein coding genes between subgroups using the DESeq R Bioconductor package. Results were analyzed using the list of miRNA targets predicted by TargetScan, enabling identification of mRNA targets that showed concordant differential expression. Finally, novel miRNAs are being identified by running miRDeep2 on these miRNA-seq samples. RESULTS: MiRNA clustering returned several distinct groups. Several represent a subset of known mRNA subgroups. For example, the MYC-amplified tumors within Group3 could be further subdivided into two separate clusters using the miRNA data. The integrated miRNA and mRNA differential expression analysis reveals some regulation patterns. For example, one of the SHH signature genes EYA1 is down-regulated in Group3 and Group4, likely because its potential regulator miR-135b is highly expressed in these tumors relative to SHH tumors. Further PAX6, an important regulator in brain development, appears down-regulated in Group3, likely because miR-96 is over-expressed in Group3 compared to others groups. CONCLUSIONS: This large-scale study reveals miRNAs with subgroup specific expression, and provides additional support for the distinct nature of the four medulloblastoma subgroups. Integration of the micronome and the transcriptome accounts for a portion of the transcriptional heterogeneity between medulloblastoma subgroups.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0079. REGULATION OF MEDULLOBLASTOMA STEM CELL SELF-RENEWAL THROUGH THE DEVELOPMENTALLY CONSERVED FoxG1-Bmi1-p21 AXIS

Branavan Manoranjan 1, Xin Wang 1, Robin Hallett 1, Chitra Venugopal 1, Stephen Mack 2, Nicole McFarlane 1, Sara Nolte 1, Katrin Scheinemann 1, Thorsteinn Gunnarsson 1, John Hassell 1, Michael Taylor 2, Cathy Lee 3, Joanna Triscott 3, Colleen Foster 3, Christopher Dunham 3, Cynthia Hawkins 2, Sandra Dunn 3, Sheila Singh 1

Abstract

INTRODUCTION: Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. By merging cancer genomics and developmental biology, recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Subgroups 1 and 2 are characterized by a relatively good clinical outcome and activation of the Wnt and Shh signaling pathways, respectively. In contrast, subgroups 3 and 4 ("non-Shh/Wnt subgroups") are distinguished by metastatic disease, poor patient outcome, and the current deficiency of a defined signaling pathway phenotype. METHOD: Gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in subgroup 3 and 4 MBs, as BTICs typically comprise a minority of cells and may therefore be represented by low transcript levels when profiling bulk MBs. Since BTIC frequency has been associated with tumor aggressiveness and poor outcome, we investigated the differential subgroup-specific stem cell gene expression patten of 325 primary human MBs. Using a subsequent series of step-wise knockdown, overexpression, in vitro, in vivo, and ChIP analyses we then assessed the functional relevance of two genes, Bmi1 and FoxG1, which were preferentially expressed in non-Shh/Wnt MBs. RESULTS: For the first time we show FoxG1 and Bmi1 to co-operatively function to regulate in vitro self-renewal and in vivo tumorigenicity. Our ChIP data demonstrates that a differential transcriptional regulatory mechanism exists between unsorted and sort-enriched stem cell populations, in which Bmi1 and FoxG1 show reciprocal promoter binding only in CD15+ MB stem cells. Our data further suggests this difference to repress cell cycle inhibitor, p21, in CD15+ MB stem cells, permitting extensive self-renewal and proliferation in this cell fraction, while CD15- cells are maintained under the control of p21. CONCLUSIONS: Our model illustrates Bmi1 as a novel downstream target of FoxG1, through which it exerts an increase in stem cell self-renewal and tumorigenicity by inhibition of p21. Through the application of stem cell data gathered from genomic platforms, we have performed functional BTIC assays to discover novel BTIC self-renewal mechanisms amenable to therapeutic targeting. We further demonstrate the presence of developmentally conserved pathways such as the FoxG1-Bmi1-p21 axis in tumor-initiating cells, indicating brain tumorigenesis as development gone awry. BTIC-specific targeted therapies involving these regulatory networks may assist in improving the overall survivorship of non-Shh/Wnt subgroup MBs patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0080. GROSS TOTAL RESECTION AND SUPRATENTORIAL LOCATION SIGNIFICANTLY IMPROVE OVERALL SURVIVAL IN PEDIATRIC PATIENTS WITH HIGH GRADE GLIOMA

Heather J McCrea 1, Evan Bander 1, Rachael A Venn 1, Anne S Reiner 1, J Bryan Iorgulescu 1, Luis A Puchi 1, Peter M Schaefer 1, Gustav Cederquist 1, Jeffrey P Greenfield 1

Abstract

INTRODUCTION: Although 50% of pediatric CNS tumors are gliomas, higher WHO grade astrocytomas are less common within this group (<10% of CNS tumors). Survival, duration, and prognostic factors in adults have been comprehensively analyzed, but less is known about factors contributing to overall survival (OS) and progression free survival (PFS) in pediatric high grade glioma. METHOD: We reviewed databases from Memorial-Sloan Kettering Cancer Center between 1988 and 2010 including patients if they were 21 years or less at time of diagnosis, had a diagnosis of glioblastoma, anaplastic astrocytoma, high grade astrocytoma, or high grade oligoastrocytoma. Brainstem gliomas were also included in the analysis if they had not undergone biopsy suggestive of a lower grade. Kaplan-Meier curves and log-rank statistics were used to compare groups univariately. Multivariate analyses were completed using Cox proportional hazards regression models. RESULTS: 102 patients were identified with median age of 11 years. Median OS was 1.9 years, and median PFS was 278 days. Location was significant (p < .0001): children with supratentorial, brainstem, thalamic, or cerebellar tumors had median overall survivals of 2.7, 1.0, 1.3, and 1.9 years, respectively. Patients with GTR had median OS of 4 years vs. 1.6 years for STR and 1.3 years for biopsy patients (P < .0009). PFS varied by age subgroups with patients≤ 3 years and those 13 and older having significantly longer PFS (P = .002). Associations remained significant in multivariate analyses. CONCLUSIONS: OS was significantly correlated with location of tumor and extent of resection. In contrast, the only variable statistically correlated with PFS was age at surgery. Neither OS nor PFS were significantly different between those treated before 2000 or after 2000. The majority of patients were treated with chemotherapy and radiation, thus analysis could not be performed to determine if certain subgroups benefited from these treatments. Retrospective molecular analysis will hopefully further clarify subgroups of patients within this difficult to treat population.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0081. TARGETING MITOCHONDRIA AND METABOLISM AS A NOVEL THERAPEUTIC APPROACH IN THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA

Maria Tsoli 1, Peter Luk 2, Pierre Dilda 2, Phil Hogg 2, Michelle Haber 1, David Ziegler 1

Abstract

INTRODUCTION: Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumours. They mostly affect young children and, as there are no effective treatments, almost all will die of their tumour within 12 months. Mitochondria are attractive cancer drug targets as they play a fundamental role in energy production through glucose and lipid metabolism as well as cell growth and apoptosis. One of the key proteins responsible for maintaining proper mitochondrial function is adenine nucleotide translocase (ANT). A recently developed ANT inhibitor, PENAO has been suggested to inhibit mitochondrial function and glucose metabolism. METHOD: To evaluate the cytotoxic effectiveness of PENAO in DIPG neurospheres a number of methodologies were employed. These included, alamar blue assays, Annexin/PI and caspase 3/7 activities. To assess loss of mitochondrial integrity we performed flow cytometric analysis of cytosolic superoxide production (dehydroethidium stain) and membrane depolarization (JC-1 stain) in PENAO-treated DIPG neurospheres. Inhibition of glucose metabolism was examined by a lactate assay. To assess whether the cytotoxic ability of PENAO can be further enhanced in DIPG neurospheres we performed combination treatments of PENAO/Reversan (MRP1 transporter inhibitor) and PENAO/3BP (glycolysis inhibitor) and examined cell death by alamar blue and Annexin/PI staining. RESULTS: Treatment of DIPG neurospheres with PENAO profoundly inhibited cellular proliferation with IC50 concentrations range of 0.5-3uM. Sensitivity to PENAO was found to be associated with increased apoptotic death and caspase 3/7 induction. Effectiveness of PENAO was found to be enhanced upon combination with MRP1 inhibitor Reversan leading to IC50s within the 0.1-0.5uM range and increased apoptotic death. Loss of mitochondrial activity was observed with increased cytosolic levels of superoxide and depolarised mitochondria. Treatment of DIPG cells with PENAO did not inhibit lactic acid production indicating that this drug does not inhibit glycolysis. Combination treatment with 3-Bromopyruvate further enhanced the cytotoxic effects of PENAO. CONCLUSIONS: These findings indicate that blocking mitochondrial ANT particularly is a promising therapeutic strategy to treat unresectable and irradiation-resistant DIPG.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0082. MYXOPAPILLARY SPINAL EPENDYMOMAS DISPLAY A HIF1-ALPHA DRIVEN ‘WARBURG’ PHENOTYPE

Stephen Mack 1, Sameer Agnihotri 1, Hendrik Witt 2, David Shih 1, Xin Wang 1, Vijay Ramaswamy 1, Kory Zayne 1, Kelsey Bertrand 1, Luca Massimi 3, Wiesia Grajkowska 4, Boleslaw Lach 5, Nalin Gupta 6, William Weiss 6, Abhijit Guha 1, Gelareh Zadeh 1, James Rutka 1, Andrey Korshunov 2, Stefan Pfister 2, Michael Taylor 1

Abstract

INTRODUCTION: Spinal ependymomas are rare slow-growing tumours found in both adults and children. Myxopapillary ependymomas are a distinct histological variant arising predominantly in the conus medullaris, cauda equina, or filum terminale. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently the only effective treatment for myxopapillary ependymoma is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy. METHOD: Gene expression profiling was performed on 35 spinal ependymomas (Affymetrix Gene 1.1ST), and copy number profiling on and overlapping cohort of 38 spinal ependymomas. (Affymetrix SNP6.0). GISTIC2.0 was used to identify significant broad and focal copy number events. Consensus hierarchical clustering and non-negative matrix factorization were used to establish subgroup assignments. Pathway analysis was performed using gene set enrichment analysis and visualized with Cytoscape: Enrichment map. Western blot analysis was used to confirm gene expression values. Functional validation experiments were performed on tumour lysate consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production. RESULTS: At a gene expression level, we demonstrate that classic and myxopapillary spinal ependymomas are molecularly and biologically distinct. These findings are supported by specific copy number alterations occurring in each histological variant. Pathway analysis revealed that myxopapillary ependymoma are characterized by increased cellular metabolism, associated with up-regulation of HIF-1a and its transcriptional targets. These findings were validated by western blot analysis demonstrating increased protein expression of HIF-1a, HK2, PDK1, and phosphorylation of PDHE1a. Functional assays were performed on myxopapillary tumour lysates to demonstrate decreased PKM activity, increased HK activity, and elevated lactate production. CONCLUSIONS: Our findings suggest that myxopapillary ependymoma may be driven by a Warburg metabolic phenotype, mediated by the HIF1a transcriptional network. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by next-generation small molecule inhibitors/activators that inhibit aerobic glycolysis, and which should be tested in pre-clinical studies as therapy for myxopapillary ependymoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0083. EFFECTIVE EPIGENETIC THERAPY FOR CIMP+ POSTERIOR FOSSA EPENDYMOMA

Stephen Mack 1, Hendrik Witt 2, Natalie Jager 2, Scott Zuyderduyn 3, Kathleen Nethery-Brokx 1, Livia Garzia 1, Kory Zayne 1, Xin Wang 1, Mark Barszczyk 1, Khalida Wani 4, Eric Bouffet 1, William Weiss 5, Cynthia Hawkins 1, James Rutka 1, Gary Bader 3, Kenneth Aldape 4, Peter Dirks 1, Stefan Pfister 2, Andrey Korshunov 2, Michael Taylor 1

Abstract

INTRODUCTION: Ependymoma is the third most common pediatric brain tumor and remains incurable in 45% of patients. We previously reported the identification of two molecularly and clinically distinct subgroups of posterior fossa (PF) ependymoma, which we named Group A and B. While patients with Group B tumors harbor a large number of gross chromosomal alterations and have favorable prognoses, patients with Group A tumors have balanced genomic profiles with poor clinical outcomes. Given the rarity of large genomic alterations, we hypothesized that SNVs or epigenetic alterations could be contributing to the pathogenesis of Group A ependymoma. METHOD: Somatic single nucleotide variants were identified by whole-genome sequencing 5 PF, and whole–exome sequencing 30 PF ependymomas. Promoter methylation profiling was performed on 79 ependymomas by MBD2-chip (Nimblegen CpG Island Promoter Plus arrays). Methylation results were validated by profiling a non-overlapping cohort of 48 PF ependymomas on a different platform (Illumina Infinium 450K). In vitro and in vivo experiments were performed on four primary ependymoma cell cultures using the DNA methylation inhibitors, Decitabine, and H3K27me3 inhibitors, 3-Dezaneplanocin A and GSK343/GSK503C. RESULTS: We determined that the genetic landscape of Group A ependymoma is remarkably silent, exhibiting no recurrent somatic SNVs. In contrast, Group A and B PF ependymoma are characterized by distinct DNA methylation profiles, and Group A ependymoma may be driven by a CpG island methylator phenotype (CIMP + ve). Pathway analysis revealed that the genes targeted by DNA hypermethylation in Group A ependymoma overlapped significantly with genes marked by the PRC2 complex in embryonic stem cells. Finally, PFA ependymoma short-term cultures were highly sensitive to pharmacologic disruption of DNA methylation and H3K27 tri-methylation by pathway inhibitors both in vitro and in vivo. CONCLUSIONS: We propose two diverse mechanisms leading to tumourigenesis in Group A and B ependymoma. The greater number of chromosomal alterations in Group B suggests a chromosomal instability phenotype (CIN), while the increased number of methylated CpG islands in Group A suggests a CpG island Methylator phenotype (CIMP). Our findings also represent the first subgroup specific therapy shown to be effective in Group A (CIMP+) ependymoma, and suggest that agents targeting DNA methylation and/or H3K27 tri-methylation may be efficacious to use in patients harboring CIMP+ PF ependymoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0084. DIVERGENT GROWTH AND INVASIVE PATTERNS IN A NEURAL PROGENITOR CELL-BASED MODEL FOR GLIOMA

Jane Engler 1, Aaron Robinson 1, Anna Wade 1, Annette Molinaro 1, Joanna Phillips 1

Abstract

INTRODUCTION: Pediatric high-grade astrocytoma (HGA) is highly invasive, driving malignant tumor cells deep within the adjacent brain. Identification of the determinants of tumor cell invasion is critical both for the design of new therapeutic strategies that target these factors and for an improved understanding of the pathways that drive this behavior in the diffuse gliomas. Interestingly, tumors can have diverse patterns of intraparenchymal invasion, including perivascular invasion and diffuse, single cell invasion. To identify the molecular mechanisms that drive different invasive patterns, we have established two murine gliomas with distinct growth and invasive phenotypes. METHOD: To model pediatric disease we isolated neural progenitor cells from the postnatal day 5 brain of Ink4a/Arf−/– mice, and transduced with hEGFRvIII. While less frequent than in the adult, EGFR alterations are present in pediatric HGA, including the constitutively active vIII variant. Intracerebral transplantation of tumor-prone neurospheres generated two tumor lines with divergent and stable phenotypes: large bulky tumors with perivascular invasion (PV) versus smaller diffuse tumors with single cell invasion (DI) (p = 0.045, n = 19). To investigate molecular mechanisms driving these phenotypes ZsGreen tagged tumor cells were sorted from brain tumors (n = 6 from each line) and transcriptionally profiled. RESULTS: PV tumors were found to overexpress genes related to DNA replication & repair and the cell cycle process (>2-fold, FDR < 1%), consistent with their higher proliferative rate in vitro (average doubling time: 20hrs versus 38hrs, p < 0.001) and shorter median survival (21d vs. 30d DI tumors, p = 0.0022). Interestingly, even at equivalent median survival time points (21d and 28d, respectively) tumor cell number was greater for PV than for DI (average: 3x107 cells versus 3.5x106 cells, p = 0.001). In contrast, DI tumors had significantly higher expression of genes associated with interactions with the extracellular environment, including cell adhesion molecules, chondroitin and heparan sulfate biosynthesis. CONCLUSIONS We have generated a neural progenitor cell-based murine model for HGA that recapitulates some of the heterogeneity of the human disease, including divergent invasive phenotype and survival. PV tumors appear to undergo a primarily proliferative drive and DI tumors show increased extracellular interactions. We are currently investigating downstream signaling pathways and key transcription factors to understand the determinants of this heterogeneity and we are investigating these determinants in human pediatric HGA. As both response to therapy and therapeutic approach may be influenced by tumor cell invasion, it is critical that we better understand this fundamental process.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0085. SUBGROUP SPECIFIC PATTERNS OF RECURRENCE IN MEDULLOBLASTOMA

Vijay Ramaswamy 1, Marc Remke 1, Eric Bouffet 1, Claudia Faria 1, David Shih 1, Sridharan Gururangan 2, Roger McLendon 2, Ulrich Schuller 3, Keith Ligon 5, Scott Pomeroy 5, Nada Jabado 6, Sandra Dunn 7, Maryam Fouladi 4, James Rutka 1, Cynthia Hawkins 1, Uri Tabori 1, Roger Packer 8, Stefan Pfister 9, Andrey Korshunov 9, Michael Taylor 1

Abstract

INTRODUCTION: Recurrent medulloblastoma remains an enormous treatment challenge, and is almost uniformly fatal. Recent studies confirmed that medulloblastoma comprises four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns. METHOD: We identified a screening cohort of all recurrent medulloblastomas at the Hospital for Sick Children between 1994-2012, and sub-grouped the cases from FFPE tissues using a nanoString-based gene expression class predication algorithm. Our findings were confirmed through analysis of two independent non-overlapping FFPE validation cohorts. RESULTS: A screening cohort of 30 recurrent medulloblastoma was assembled; 9 local tumor bed recurrences, 21 metastatic recurrences. SHH tumors recurred more frequently in the tumor bed (8/11, 73%), Group 3 and Group 4 recur more exclusively with metastases (16/18, 89%; p < 0.001). Latency to death post recurrence was longer in Group 4 patients (p = 0.03). The subgroup-specific location of recurrence was confirmed in a multicenter cohort (p = 0.02, n = 40), and an independent cohort (SHH 21/24 local recurrences, Group 3/4 69/72 metastatic relapses, n = 96, p < 0.001). Strikingly, in all 40 cases where matched primary and recurrent pairs were available, subgroup affiliation remained stable at recurrence. CONCLUSIONS: Medulloblastoma does not change subgroup at the time of recurrence. Significant differences in the pattern of recurrence exist across medulloblastoma subgroups, further highlighting the clinical differences between the four principle subgroups. Intensified local (posterior fossa) therapy should be considered upon initial treatment for SHH patients. Refinement of therapy for Groups 3 and 4 should focus on the metastatic compartment.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0086. EFFECTIVE TARGETING OF THE HGF/c-MET PATHWAY IN MEDULLOBLASTOMA

Claudia Faria 1, Adrian Dubuc 1, Brian Golbourn 1, Roberto Diaz 1, Sameer Agnihotri 1, Nesrin Sabha 1, Amanda Luck 1, Michael Leadly 2, Denis Reynaud 2, Xiaochong Wu 1, Marc Remke 1, Vijay Ramaswamy 1, Paul Northcott 3, Stefan Pfister 3, Sidney Croul 4, Marcel Kool 3, Andrey Korshunov 5, Christian Smith 1, Michael Taylor 1, James Rutka 1

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor in childhood and accounts for approximately 10% of all pediatric cancer deaths. We previously reported the importance of the hepatocyte growth factor (HGF)/c-MET signaling pathway in the pathogenesis of medulloblastoma. High expression of c-MET is found most often in conjunction with the sonic hedgehog (SHH) and some Group 3 medulloblastomas. We hypothesize that subgroup-specific targeting of c-MET could be effective therapy in medulloblastoma. METHOD: We performed mRNA expression analysis of c-MET in a cohort of 103 medulloblastomas and confirmed the results using a non-overlapping validation cohort of 439 medulloblastomas. Immunostaining of clinically annotated medulloblastoma tissue microarrays with c-MET and phospho-c-MET antibodies was performed, and the expression levels were correlated with survival. Foretinib, an orally available multikinase inhibitor of c-MET, was used for all in vitro and in vivo experiments. Two cell lines representative of SHH and Group 3 tumors were used. To determine the ability of foretinib to penetrate the blood-brain barrier we used high-performance liquid chromatography with mass spectrometry detection. RESULTS: We determined that c-MET is highly expressed, both at the transcriptional and at the protein level, in SHH medulloblastomas (81%) and some Group 3 tumors (33%). In the SHH subgroup, high expression of c-MET and phospho-c-MET (the activated receptor) correlates with a shorter progression free survival. We showed that foretinib inhibits proliferation, migration and invasion of medulloblastoma cell lines in a dose-dependent manner. We also determined that foretinib crosses the blood-brain barrier and can be quantified in the mouse brain. In medulloblastoma xenografts, foretinib was able to reduce tumor growth and metastasis and to increase survival when compared to controls. CONCLUSIONS: The HGF/c-MET is a targetable pathway in medulloblastoma. Foretinib is an orally available inhibitor of c-MET that crosses the blood-brain barrier, shows antitumor effect and prolongs survival in preclinical models of medulloblastoma. Our findings suggest that a subgroup of patients harbouring high c-MET medulloblastomas may benefit from targeted therapies with c-MET inhibitors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0087. CHROMOSOME 1q GAIN AS GENETIC MARKER FOR RISK STRATIFICATION OF PEDIATRIC EPENDYMOMA PATIENTS - RESULTS FROM THE GERMAN MULTICENTER HIT2000 TRIAL

Torsten Pietsch 1, Evelyn Doerner 1, Anja zur Muehlen 1, Natalia Velez-Char 1, Monika Warmuth-Metz 2, Rolf Kortmann 3, Katja von Hoff 4, Carsten Friedrich 4, Stefan Rutkowski 4, Andre von Bueren 4

Abstract

INTRODUCTION: In contrast to histopathological grading which varied in respect to its prognostic value between different clinical trials, extent of resection was found to be consistently associated to the clinical outcome. In retrospective series, gain of genetic material of chromosome arm 1q was identified to predict worse outcome. This marker was mainly assessed by FISH analysis which typically showed failure rates of 15–20 % in archival material. METHOD: To validate this marker in a homogenously treated patient cohort, we analysed chromosome 1q in 209 consecutive cases enrolled into the multicenter trial HIT2000 (postoperative chemotherapy and irradiation, in a sequence depending on age and extent of resection) in which formalin-fixed, paraffin embedded material was available for DNA extraction. By using multiplex ligation-dependent probe amplification (MLPA) for 5 markers located on chromosome 1q and control markers, we were able to analyse 206/209 samples (>98 %). RESULTS: We found gain of chromosome 1q in 35 cases (17%). Interestingly, only 2 of 35 WHO grade II ependymomas had 1q gain (5.7%). 35 cases were diagnosed as WHO grade II ependymoma, 171 as anaplastic ependymoma (WHO grade III). At a median follow-up of 3.9 years for survivors, patients with tumors showing 1q gain had a significantly lower 4-year overall survival (OS) (±SE) of 58% +/- 9% compared to patients lacking this marker (89% +/-3%, p = 0.002). Multivariable analysis demonstrated that residual tumor and infratentorial localization were independent risk factors for event-free survival, and gain of chromosome 1q for OS, respectively. CONCLUSIONS: In conclusion, we validated chromosomal 1q gain to be a useful independent genetic marker for risk stratification of pediatric ependymoma patients which can be evaluated by MLPA representing a robust, reliable and cost-efficient method.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0088. ESTABLISHMENT OF DIFFUSE PONTINE GLIOMA CELL LINES AND XENOGRAFTS

Yu-Jen Lu 1,2, C David James 1, Rintaro Hashizume 1, Sabine Mueller 1, Joanna Phillips 1, Nalin Gupta 1

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is an infrequent but uniformly fatal cancer that most often affects children under 5 years of age. As a result there is an urgent need to identify novel therapies to improve patient outcomes. Because tissue is rarely obtained at diagnosis, there is a dearth of preclinical resources and models that would enable study of the molecular biology of DIPG, the information from which could be used for therapeutic hypothesis testing. The purpose of our ongoing investigation is to assess the feasibility of developing sustainable cell resources obtained from DIPG patients. METHOD: Patients' guardians provided consent through an approved tissue acquisition protocol. A frameless stereotactic system was used to select the target and trajectory through the cerebellar hemisphere and peduncle, in 5 pediatric patients, ages 3-10. A side-biting needle was used to obtain small tissue cores (1x4 mm) for confirmation by histopathology, and also for adhesion and/or neurosphere cell culture establishment. In one case, cells were transduced with a retrovirus vector containing human telomerase reverse transcriptase (hTERT) for cell immortalization. Cell cultures showing sustainable growth were transduced with a lentiviral vector containing firefly luciferase (Fluc) for in vivo bioluminescence imaging. RESULTS: Five DIPG tissues have been obtained by surgical biopsy, with a sixth specimen obtained at autopsy. Of the six samples in total, two have yielded DIPG cell lines. One of these cell lines, however, required hTERT modification for sustainable cell culture growth. Both of the DIPG cell lines produce brainstem tumors in athymic rodents that recapitulate many of the features of the human disease, including a highly infiltrative pattern of growth. Luciferase modification of each of DIPG cell line has resulted in derivatives that are useful for monitoring orthotopic tumor growth and response to therapy by bioluminescence imaging. CONCLUSIONS: Excluding modifications, one of five surgical DIPG specimens has generated a sustainable cell line, and this cell line is tumorigenic, expanding its utility to xenograft-associated research. hTERT modification of another DIPG specimen resulted in its sustainable propagation, with derivative cells capable of forming tumors in athymic mice. Though preliminary, our experience to date supports the feasibility of developing a panel of DIPG cell lines and xenografts, though such an initiative would likely benefit from a multi-institutional approach that would take advantage of an increased number of opportunities for establishing DIPG resources.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0089. MOLECULAR PROFILING REVEALS CNS-PNETS OVERLAP WITH VARIOUS WELL-DEFINED ENTITIES

Dominik Sturm 1, Paul A Northcott 1, David TW Jones 1, Andrey Korshunov 2, Daniel Picard 3, Peter Lichter 4, Annie Huang 3, Stefan M Pfister 1, Marcel Kool 1

Abstract

INTRODUCTION: Childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are poorly classified embryonal tumors with early onset and aggressive clinical behavior. Histological diagnosis is contentious and complicated by divergent differentiation along neuronal, astrocytic, or other lines and is based on absence of a clear morphological pattern. In recent profiling studies, we identified molecular subgroups of CNS-PNETs with biological characteristics suggestive of overlap with other childhood CNS tumors. Here, we aimed at a comprehensive molecular characterization of CNS-PNET in relation to a large cohort of other classes of childhood brain tumors to further define/clarify the molecular nature of tumors diagnosed as CNS-PNETs. METHOD: A large cohort of 147 fresh frozen or paraffin-embedded tumor samples with the institutional diagnosis "CNS-PNET" was investigated for genome-wide DNA methylation patterns and copy-number aberrations using Illumina Infinium HumanMethylation450K Arrays. Transcriptomic profiling on Affymetrix GeneChip U133 Plus2.0 Arrays was performed for a subset of cases (n = 45). Molecular profiles of selected cases were complemented by targeted sequencing of H3F3A and IDH1, immunostaining for INI1, LIN28A, and OLIG2, and Fluorescence In Situ Hybridization for 19q13.42. "CNS-PNET" profiles were compared to the molecular genetic make-up of >500 other childhood brain tumors including medulloblastomas, pilocytic astrocytomas, ependymomas, glioblastomas, AT/RTs, and ETMRs (ETANTRs). RESULTS: While unsupervised clustering methods of DNA methylation and gene expression profiles clearly segregated most tumor types by histologic entity and molecular subgroup, tumors classified as "CNS-PNET" did not form a separate cluster, but were scattered across the entire spectrum of entities. Molecular profiles of CNS-PNETs displaying focal amplification of 19q13.42 and high expression of LIN28A (7/147) overlapped with ETMRs, while loss of 22q and absence of INI1 expression (6/147) were seen in CNS-PNETs clustered with AT/RTs. Mutations in H3F3A (5/147) or IDH1 (5/147) were confirmed in CNS-PNETs with DNA methylation and transcriptomic profiles matching those of glioblastomas. CONCLUSIONS: The correct classification of CNS-PNET remains difficult. Of 147 tumors with an institutional diagnosis "CNS-PNET", many cases were reliably re-classified as ETMRs, AT/RTs, or glioblastomas based on recurrent genetic aberrations. Others resembled misdiagnosed medulloblastomas or supratentorial ependymomas. Genome-wide profiling suggests that a significant proportion of CNS-PNETs diagnosed by current criteria may have overlapping molecular features with or comprise a variety of other tumor subtypes. Deeper molecular analysis, central histopathological re-evaluation of HE-stained sections, and comparison with large-scale datasets of other tumors will reveal whether the group of "CNS-PNETs", vaguely defined by histology, can essentially be reassigned to existing, well-defined entities.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0090. EVALUATING HISTONE DEACETYLASE INHIBITOR SENSITIVITY IN THREE-DIMENSIONAL MODELS OF PEDIATRIC EPENDYMOMA

Jennifer Ward 1, Catherine Teague 1, Bharati Shriyan 1, Richard Grundy 1, Ruman Rahman 1

Abstract

INTRODUCTION: Histone deacetylases are known to aberrantly regulate oncogenesis through remodeling of chromatin, resulting in gene repression. We have previously shown that histone deacetylase inhibition (HDACi) results in proliferation impairment and apoptosis of childhood brain tumor cells. However, the mechanism of HDACi remains elusive with no predictive biomarker available as an HDACi sensitivity determinant in brain tumors. Here we utilize a dynamic in vitro three-dimensional (3D) model to determine sensitivity of ependymoma cells to a panel of histone deacetylase inhibitors and evaluate expression of the proteasomal component HR23B as an HDACi putative biomarker of response. METHOD: We cultured Res196 and BXD1425 ependymoma cells as 3D aggregates using the Rotary Cell Culture System (RCCS) and compared data to 2D monolayer cultures. Ependymoma aggregates were characterized by immunohistochemistry and proliferation rates measured by the Alamar Blue proliferation assay. To determine HDACi sensitivity, Res196 and BXD1425 cells were exposed to vorinostat, entinostat and sodium valproate in 2D and 3D RCCS cultures. TRAP assay was used to determine inhibition of telomerase activity in this context. To determine if HR23B is directly associated with HDACi sensitivity, siRNA was used to knock-down HR23B prior to histone deacetylase inhibitor exposure. RESULTS: RCCS ependymoma aggregates better recapitulate the morphologic heterogeneity of primary ependymoma, express markers of tumor-derived vascular channels (processes not observed in 2D) and exhibit slower proliferation rates (11-fold for Res196; 1.25-fold for BXD1425). Res196 and BXD1425 were sensitive to vorinostat (IC50 1.5uM, 8.0uM respectively) and entinostat (IC50 3.0uM, 4.0uM respectively) upon exposure in 2D culture; however, sensitivity was 6-10-fold reduced in 3D culture. siRNA targeting of HR23B resulted in ∼90% knockdown, allowing us to determine whether HR23B is associated with HDACi sensitivity. However HR23B protein expression was not prognostic of either overall or progression-free survival in a prospective ependymoma cohort. CONCLUSIONS: Sensitivity to HDACi is significantly reduced in RCCS 3D culture compared to conventional 2D cultures, likely reflecting impaired drug penetration into tumor aggregates and drug resistance induced by the tumor microenvironment. We propose that 3D culture provides a better approximation of in vivo and clinical responses. Studies to determine whether telomerase inhibition is a mechanism of HDACi, whether HR23B is required for sensitivity to HDACi and whether ependymoma cells are sensitive to the sodium valproate will be discussed.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0091. THE GENOMIC LANDSCAPE OF TREATMENT-NAÏVE DIPG BIOPSY SAMPLES

Katy Taylor 1, Alan Mackay 1, Olena Morozova 2, Yarron Butterfield 2, Nathalène Truffaux 3, Cathy Philippe 3, Mara Vinci 1, Carmen de Torres 4, Ofelia Cruz 4, Jaume Mora 4, Darren Hargrave 5, Stéphanie Puget 6, Stephen Yip 2, Chris Jones 1, Jacques Grill 3

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) have a universally dismal prognosis (median 9-12 months), with neither chemotherapeutic or targeted agents showing any substantial survival benefit in clinical trials in children with this disease. Recent high-throughput sequencing approaches have revealed a striking prevalence of K27M mutations in the genes encoding the histone variants H3.3 (H3F3A) or H3.1 (HIST1H3B), but little has been reported of the secondary mutations accompanying these changes, nor of structural or non-coding variants. In addition, clinical practice has necessitated the study of samples retrieved from autopsy, and it is unclear whether the mutational spectrum is secondary to treatment-related changes. METHOD: We have carried out whole genome sequencing of 20 DIPG samples taken at diagnosis by stereotactic biopsy at The Necker Hospital, Paris, which were confirmed histologically as high grade astrocytomas. Raw sequence reads from tumour and matched constitutive DNA were aligned to the reference genome and analysed using a somatic analysis pipeline to identify single nucleotide variants (SNV), small insertions and deletions (Indels) and large structural variants (SV). A median of 30Gb of sequence was generated for each genome representing a median depth of >40x corresponding to a coverage of greater than 10x across 95% of known genes. RESULTS: Interim analysis has shown the number of somatic coding variants in DIPG biopsies to range from 8-22 (mean = 16) per sample, representing a frequency of 0.5-1.7 mutations per Mb. This is similar to that observed in supratentorial paediatric (3-31, mean = 15) but significantly lower than adult GBM (17-83, mean = 47), as well as a small series of DIPG autopsy samples (6-40, mean = 24, mutation frequency = 1.3-4.6/Mb). Although the transition/transversion ratios were similar between biopsy and autopsy cases, the sequence context differed, with C > T/G > A transitions more likely to be proceeded by a C and succeeded by a G in untreated than treated samples. CONCLUSIONS: K27M mutations were observed in all cases, usually in conjunction with TP53 (75%), and infrequently ATRX/DAXX. Secondary to the histone mutations, multiple mechanisms of common pathway dysregulation are observed, e.g. independent SNVs and Indels in PIK3R1 and copy number amplification of receptor tyrosine kinases. Differences in the mutational spectrum and sequence context between untreated biopsy and treated autopsy samples suggests that the increased number of mutations found in the latter may involve changes associated with clonal evolution. Study of the DIPG genome at biopsy provide a unique opportunity to identify targets for novel intervention in the upfront setting.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0092. VEGF/FGF-DEPENDENT VASCULOGENIC MIMICRY AND TUMOR-DERIVED ANGIOGENIC RESPONSE IN HIGH GRADE GLIOMA

Stuart Smith 1, Jennifer Ward 1, Christopher Tan 1, Richard Grundy 1, Ruman Rahman 1

Abstract

INTRODUCTION: The assumption of a genetically stable endothelial phenotype has been challenged with evidence of tumor-derived endothelial cells (TDEC) in gliomas. TDEC are phenotypically diverse from recruited endothelial cells and can form tumor vessels that derive from an embryonic-like process intrinsic to the tumor cell, termed ‘vasculogenic mimicry' (VM). Whether glioma cells merely mimic the vessel function or whether glioma stem-like cells transdifferentiate into endothelial cells is unclear Here we investigate whether VM and the glioma-derived angiogenic response can be modeled and manipulated in a dynamic 3-dimensional (3D) brain tumor culture by virtue of response to microenvironmental stimuli such as hypoxia. METHOD: We cultured glioblastoma, ependymoma, medulloblastoma, PNET and untransformed neural cells as 3D aggregates using the Rotary Cell Culture System (RCCS) in addition to brain tumor/brain endothelia co-cultures. Tumor-derived angiogenic response was analyzed by array qRT-PCR of 84 angiogenesis-related genes and endothelial protein expression analyzed by immunohistochemistry / immunofluorescence. Data was compared to angiogenic expression in primary glioma explants cultured in 3D, primary high grade glioma tissue and 2D cultures. Mouse flank xenografts were used to confirm the prevalence of TDEC in vivo. Small molecule competitive inhibitors of VEGF/FGF receptors were used in attempting to abrogate VM and attenuate the tumor-derived angiogenic response. RESULTS: A 3D-induced microenvironment is sufficient to promote expression of the endothelial markers CD105, CD31 and vWF in a proportion of high grade glioma, ependymoma and PNET cells, but was absent in medulloblastoma and neural stem cells. Co-cultures with brain endothelial cells demonstrated comparable CD105 expression, whereas CD31 expression was absent. Glioma xenografts reveal CD105/CD31 positive vessel-like structures near necrotic areas and TDEC CD105/CD31 expression was widespread in primary glioma tissue. Many pro-angiogenic genes were upregulated in glioma aggregates and primary glioma explants. Inhibition of either VEGF or FGF signaling was sufficient to impair VM and downregulate the tumor-derived angiogenic response. CONCLUSIONS: The RCCS permits VM modeling, allowing manipulation of this potential anti-angiogenic therapy resistance mechanism. Our findings support studies indicating that hypoxia promotes the microvascular niche whereby TDEC may derive from a tumor stem-like cell. The prevalence of TDEC expression and VM structures in xenografts and primary glioma tissue indicates that these are distinct biological processes contributing to neo-vascularization in primary gliomas. Co-culture experiments suggest that in the presence of sufficient pre-existing endothelial cells there is less selective pressure for TDEC to form. VEGF and FGF inhibition using small molecule inhibitors are promising strategies for reducing angiogenic response and inhibiting VM.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0093. HISTONE H3.3 MUTATIONS DRIVE PAEDIATRIC GLIOBLASTOMA THROUGH UPREGULATION OF MYCN

Lynn Bjerke 1, Alan Mackay 1, Meera Nandhabalan 1, Anna Burford 1, Alexa Jury 1, Sergey Popov 1, Dorine Bax 1, Diana Carvalho 1, Katy Taylor 1, Mara Vinci 1, Ilirjana Bajrami 1, Imelda McGonnell 2, Chris Lord 1, Rui Reis 4, Darren Hargrave 3, Alan Ashworth 1, Paul Workman 1, Chris Jones 1

Abstract

INTRODUCTION: Glioblastomas of children and young adults (pGBM) have a median survival of only 12-15 months and are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant (H3F3A), occurring either at or close to key residues marked by methylation for regulation of transcription - K27 and G34. Although the recurrent base changes may be predicted to cause profound effects on gene expression, the mechanism by which the mutations effect these changes and promote tumorigenesis is unknown. METHOD: Gene expression profiles of pGBM samples from independent studies were analysed on the basis of their H3F3A status. We carried out Western blot analysis and chromatin immunoprecipitation linked to whole genome sequencing (ChIP-Seq) for the histone H3K36 trimethylation mark (H3K36me3) in a panel of pGBM cell lines including the G34V mutant KNS42 cells. siRNA screening was also carried out in this panel using a library directed against the human kinome, with hits selectively targeting the G34 mutant cells validated by individual oligonucleotides and small molecule inhibition in vitro. RESULTS: We observed no difference in total H3K36me3 levels in G34 mutant versus wild-type cells, however ChIP-Seq identified 156 differentially bound and expressed genes. The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. A synthetic lethality siRNA screen revealed this driving aberration to be selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein such as AURKA and CHK1. CONCLUSIONS: The H3F3A G34 mutation is specific to tumours of the cerebral hemispheres and is associated with a distinct age of incidence and gene expression signature compared to K27 and wild-type tumours. Our data provide the mechanistic explanation for how the G34 mutation acts to deliver MYCN, a potent tumorigenic initiator, into a stem cell compartment of the developing forebrain, selectively giving rise to cerebral hemispheric glioblastoma. Employing synthetic lethal approaches to these mutant tumour cells provides a rational way to develop novel and highly selective treatment strategies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0094. INTEGRATED WHOLE GENOME AND RNA SEQUENCING IDENTIFIES NOVEL EXPRESSED FUSION TRANSCRIPTS IN PAEDIATRIC HIGH GRADE GLIOMA

Diana Carvalho 1, Alan Mackay 1, Anna Burford 1, Lynn Bjerke 1, Lina Chen 1, Iwanka Kozarewa 1, Chris Lord 1, Alan Ashworth 1, Darren Hargrave 2, Rui Reis 3, Chris Jones 1

Abstract

INTRODUCTION: Chromosomal rearrangements resulting in novel fusion genes are strongly associated with cancer and numerous examples exist in both adult and childhood malignancies. Structural variants frequently result in chimeric proteins targetable by novel therapeutic approaches, an outcome desperately needed in paediatric high grade glioma (pHGG). These tumours are increasingly understood to be driven by specific mutations in the genes encoding the histone H3.3 variant (H3F3A) and the chaperones ATRX/DAXX, however to date there have been no reports of structural variation. We sought to address this by carrying out integrated analysis of whole genome and transcriptome sequencing of three pHGG cell lines. METHOD: We fully sequenced the genomes and transcriptomes of the pHGG cell lines KNS42 (H3F3A G34V, TP53 R342*), SF188 (TP53 G266E) and UW479 (TP53 R158L, DAXX S683Y) using the IlluminaHiSeq2000 platform at 37-40x (DNA) and generating 74-92 million reads (RNA), respectively. Structural variants were detected using an integration of computed copy number and BreakDancer and ChimeraScan pipelines, and validated in the reference cells by PCR/Sanger sequencing. Break-part and/or fusion FISH probes were generated for validated expressed fusion transcripts and screened on tissue microarrays containing 130 pHGG samples to look for recurrence in clinical specimens, and the functions of these fusions assessed. RESULTS: 305 DNA-level structural variants involving one gene at either end were nominated in the three pHGG cell lines, many involving genes that are co-amplified. Several genome breakpoints junctions found showed microhomology regions (1-4bp), which are characteristic of microhomology-mediated break-induced-replication (MMBIR). SF188 harboured a complex amplified sequence including the genes PDGFRA (4q12), MYC (8q24.1), CCND1 (11q13.3), MLL (11q23.3) and CDK4 (12q14.1) found to be present as an extra-chromosomal double minute. Overlay of genome with transcriptome data identified 26 expressed fusion transcripts associated with DNA-level breakpoints. These included TULP4:RPTOR (t6;17 - SF188), GORASP2:CDADC1 (t2;13 - KNS42) and AKAP6:NUBPL (t12;14 - UW479). CONCLUSIONS: Numerous expressed fusion transcripts have been identified in pHGG cells which confer dysregulation of a variety of known actionable targets in cancer-related signalling pathways. Although the prevalence of fusions in clinical pHGG is not suggestive of structural variation driving tumorigenesis in this disease, the generation of these fusions may be a complementary mechanism to pathway dysregulation (such as PI3K/mTOR). Integration with functional screening data may help to identify novel drug targets, such as the protein kinase CDADC1, knockdown of which selectively kills pHGG cells in vitro.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0095. ENHANCING AUTOPHAGY AS STRATEGY TO TARGET HIGH GRADE GLIOMAS AND DIFFUSE INTRINSIC PONTINE GLIOMAS

Miguel Marigil 1, Patricia Jauregui Jauregui 1, Marta Alonso 1

Abstract

INTRODUCTION: Pediatric high-grade gliomas (pHGG) including diffuse intrinsic pontine gliomas (DIPG) are one of the most formidable challenges faced by pediatric oncologist. Recently the used of molecular genetics has allowed to gain a deeper understanding of the genomic make up of these tumors. IGF1R, a receptor tyrosine kinase, has been found to be amplified. Oncolytic adenoviruses engineered to replicate in and destroy tumor cells selectively represent a promising new therapeutic strategy that could improve the outcome of pHGG and DIPGs. The objective of this work is to evaluate the combined antitumor effect of an IGF1R inhibitor and the oncolytic adenovirus Delta-24-RGD. METHOD: In vitro citotoxic effect of IGF1R inhbitor was assessed in a panel of pHGG cell lines (n = 4), DIPGs cell lines (n = 2) by MTT. Assessment of viral replication was performed using the TCID50 assay. Autophagy was determined by flow cytometry using acridine orange staining. In addition, we performed western blot analyses to assess the expression of different autophagy markers such as beclin, ATG5 and LC3 lipidation and IGFR1 signalling pathway before and after the treatment. Cell cycle was evaluated by flow cytometry. Survival studies in animal models are in progress. RESULTS: Our results showed that Delta-24-RGD alone was very efficient targeting pHGGs and DIPGs cell lines, IC50s ranging from 1.5 to 5 MOIs. IGF1R alone was able to kill the pHGG cell lines with IC50s in the nanomolar or low micromolar range. Combination of the IGF1R inhibitor with Delta-24-RGD resulted in a synergistic antiglioma effect. Cell cycle analysis showed that combination-treated cells did not arrest and progressed through the cell cycle to finally die by autophagic cell death. At the moment we are testing in vivo the efficacy of this combination using ortotopic models of pHGG and DIPG in nude mice. CONCLUSIONS: Altogether our data show that Delta-24-RGD in combination with an IGF1R inhibitor induces a potent antitumor effect in these tumors through autophagic cell death. This combination could constitute a promising therapeutic approach for pHGGs and DIPGs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0096. TSP-1 MODULATES P13K SIGNALING TO ALTER METASTATIC PHENOTYPES AND CHEMO- AND RADIO-SENSITIVITY IN MEDULLOBLASTOMA

Tiffany SY Chan 1, Cynthia Hawkins 1, Daniel Picard 1, Jack Henkin 2, Annie Huang 1

Abstract

INTRODUCTION: Survival of localized medulloblastoma (MB) has improved significantly with intensified chemo-radiotherapeutic regimens in recent years, however, treatment and/or prevention of craniospinal metastases remains a major obstacle in clinical management of MB. In prior studies, we generated MYC driven xenograft models of metastatic MB and identified thrombospondin-1 (TSP-1), a potent tumour suppressor and anti-angiogenic agent, as a candidate effector and therapeutic targets in metastatic MB. In this study we sought to determine whether expression of TSP-1 or TSP-1 peptidomimetics alters metastatic behavior and chemo-radiotherapeutic response of MB in vitro and in vivo. METHOD: TSP-1 effects on MB cell growth in vivo was assessed using orthotopic xenograft assays in nu/nu mice; full brain and spine histological exam was performed to evaluate for tumor invasion and metastasis. For in vitro phenotypic assays, cell lines with stable TSP-1 expression or cells treated with a peptidomimetic to the structural homology repeat domain type 1 of TSP-1, were characterized using MTT/cell count/ Cell Death and Boyden chamber assays. For chemo and radio-sensitization assays, cells were pre-treated with TSP-1 mimetics prior to cellular and western blot assays to determine TSP-1 associated signaling pathways. RESULTS: Stable TSP-1 expression significantly impaired tumour growth and metastases in orthotopic xenograft assays; mice with TSP-1 expressing xenograft had significantly longer survival; (p < 0.02) and diminished frequency of metastases (p < 0.05). TSP-1 stable expression or mimetics treatment (50-10uM) both potently inhibited migration (decrease by up to 80%; p < 0.05) of MB cell lines with ectopic (UW426-Myc, DAOY-Myc) and high endogenous (D341, D458) Myc expression. TSP-1 mimetics treatment alone had minimal effects on MB cell proliferation/apoptosis, however, chemotherapy (Etoposide) and radiation induced MB cell death was significantly increased with TSP-1 mimetic treatment. Interestingly, TSP-1-induced phenotypic changes correlated with diminished phospho-AKT but not phospho-Erk expression. CONCLUSIONS: TSP-1 expression mediates potent effects on invasive and metastatic phenotypes in MB via inhibition of PI3K signaling. TSP-1 effects in vitro are strikingly re-capitulated by a peptide-mimetic of TSP-1 thus highlighting such mimetics as important new therapeutics for Myc-associated MB, one of the most lethal of MB variants.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0097. THE NATURE OF CHROMOSOMAL ABERRATIONS DETECTED IN PEDIATRIC PATIENTS WITH HETEROZYGOUS GERM-LINE MUTATIONS IN THE NBN GENE AND CLASSIC TYPE OF MEDULLOBLASTOMA

Joanna Trubicka 1, Marzena Kucharczyk 1, Magdalena Pelc 1, Krystyna Chrzanowska 1, Elzbieta Ciara 1, Marta Perek-Polnik 1, Wieslawa Grajkowska 1, Dorota Piekutowska-Abramczuk 1, Dorota Jurkiewicz 1, Sylwia Luczak 1, Maria Borucka-Mankiewicz 1, Pawel Kowalski 1, Małgorzata Krajewska-Walasek 1

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor in children. At present, multiple molecular dysfunctions are known to be responsible for medulloblastoma formation. Based on the comprehensive analysis four molecular subtypes of this type of cancer are identified. Nonetheless, the recent studies evaluating the role of mutations in DNA repair genes in the development of medulloblastoma indicate that changes within NBN seem to be particularly important. The aim of our study was to evaluate the nature of the chromosomal aberrations in pediatric patients with heterozygous germ-line mutations (c.511A > G and c.657_661del5) in the NBN gene with the classic type of medulloblastoma. METHOD: To identify the presence of NBN mutations direct sequencing of selected exons was performed using a 3130 Genetic Analyzer (Applied Biosystems). The analyzed sequence fragments were compared with the NBN cDNA(Gene Bank RefSeq: NC_000008.9). To determine the chromosomal aberrations we analyzed five NBN positive medulloblastoma tumor samples using a whole-genome oligonucleotide CGH microarray (NimbleGen HG18_CGX Cytogenetic 6x315K array). Feature extraction and primary data analysis were performed using Roche NimbleGen's DEVA v.1.1 software. Detailed analysis and data visualization were performed using Genoglyphix genome browser software. RESULTS: All identified in our study chromosomal aberrations have been frequently detected in medulloblastoma. In three patients with c.657_661del5 mutation we detected the monosomy or duplication of chromosome X as well as monosomy of chromosome 6. In two tumors samples from patients with c.511A > G mutation we identified the isochromosome 17q or deletion of 11p11.2-15.5, 46,XY.arr 11p11.2(195983-46386376)x1 and duplication of 11q24.1-q25, 46,XY.arr 11q24.1(122645559-134434130)x3. The obtained results allowed to prepare the preliminary correlation between the detected changes and the molecular subtype of medulloblastoma, necessary to provide improved criteria for predicting outcome for the NBN mutation carriers with this type of cancer. CONCLUSIONS: This was the first study that evaluated the pattern of chromosomal aberrations in germ-line NBN mutation carriers with medulloblastoma. The most of the identified changes were frequently altered in intermediate or poor-risk medulloblastoma (subtype C and D). In one patient we detected monosomy of chromosome 6 associated with low-risk medulloblastoma (subtype A/WNT). Our results were consistent with the patients clinical data. A comparison of clinical features between medulloblastoma patients with and without NBN mutations indicated a more aggressive course of disease in NBN heterozygotes. The study was financed by the National Science Centre, project no. 6917/B/P01/2011/40 and the Internal Project of The Children's Memorial Health Institute no S112/2009.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0099. CLINICAL AND BIOLOGIC CHARACTERISTICS OF CLEAR CELL EPENDYMOMA

Rebecca M Loret de Mola 1, Joseph Laskowski 1, Jason Fangusaro 1, Fabricio F Costa 2, Elio F Vanin 2, Stewart Goldman 1, Marcelo Bento Soares 2, Rishi R Lulla 1

Abstract

INTRODUCTION: Clear cell ependymoma (CCE) is an uncommon histologic variant of ependymoma. Additionally, CCE is clinically more aggressive with a tendency to recur and metastasize despite standard therapy. No data is available to describe the biologic differences between CCE and other ependymomas. MicroRNAs (miRNAs) are small non-coding RNA molecules that have a role in a variety of biological processes. Altered expression of miRNAs contributes to the development pediatric brain tumors including ependymoma. We aim to describe the clinical features of patients with CCE at our institution as well as identify biologic differences in miRNA expression in CCE compared to other ependymoma subtypes. METHOD: Tumor specimens and clinico-pathologic information for patients with CCE or ependymoma with clear cell features was obtained. RNA purification from tumor samples will be performed using RecoverAllTM Total Nucleic Acid Isolation system. TaqMan® miRNA assays will be used to quantify the levels of approximately 762 mature miRNAs from each sample using the Applied Biosystems 7900HT Fast Real-Time PCR system in 384-well low density arrays (TLDAs). Cycle threshold (Ct) values under 35 will be considered positive for the presence of target miRNAs. Data from miRNA profiling of CCE will be compared to other ependymoma subtypes using the RealTime StatMiner® software. RESULTS: Eleven patients with CCE or ependymoma with clear cell features were identified from a total of 166 ependymoma cases over a 20 year period. Median age at diagnosis of CCE was 7 years (range 2-16). Six patients were female (54%) and a majority of tumors were supratentorial (72%). Tumor recurrence occurred in 7 patients (63%) and 2 patients (18%) developed extraneural metastasis. Median time to recurrence was 8 months (range 7-48 months). Three patients (27%) died of their disease at the time of data censoring. MicroRNA profiling of CCE specimens is underway and will be presented. CONCLUSIONS: Our results confirm that CCE is an aggressive variant of ependymoma characterized by frequent tumor recurrence and extraneural metastases. We hypothesize that CCE is a biologically distinct disease and that the miRNA profiles of CCE patients will differ from that of other subtypes of ependymoma. Future studies include identifying changes in miRNA expression profiles over time in patients with CCE and multiple recurrences.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0100. CLONAL EVOLUTION OF MEDULLOBLASTOMA BRAIN TUMOUR-INITIATING CELLS (BTICS) IN RESPONSE TO THERAPY: DISCOVERING THE REFRACTORY BTIC POPULATION

Aneet Mann 1, Chitra Venugopal 1, Parvez Vora 1, Mohini Singh 1, Randy van Ommeren 1, Nicole McFarlane 1, Branavan Manoranjan 1, Maleeha Qazi 1, Katrin Scheinemann 1, Paula MacDonald 1, Kathy Delaney 1, Anthony Whitton 1, Sandra Dunn 2, Sheila Singh 1

Abstract

INTRODUCTION: The most clinically compelling component of the Cancer Stem Cell (CSC) hypothesis holds that CSCs evade current therapy, and are responsible for disease recurrence. Although there is strong evidence that tumor-initiating cell (TIC) populations are chemo- and radio-resistant, no studies prospectively demonstrate these treatment-resistant TICs to be exclusively causative in solid tumor relapse and recurrence. In the present study, we aim to identify the treatment-refractory brain tumor-initiating cell (BTIC) in medulloblastoma (MB), the most frequent malignant childhood brain tumor. METHOD: Human MB BTICs cultured as tumorspheres were treated with chemotherapy and radiation, and subjected to flow analysis, and in vitro stem cell assays for self-renewal, proliferation and differentiation. We developed a mouse-adapted MB therapy model using our human-mouse BTIC xenograft, in which Daoy-GFP+ or Med8a-GFP+ MB cells were intracranially transplanted into NOD-SCID mice that were then treated with radiation, and chemotherapeutic drugs Vincristine, Cisplatin and Cyclophosphamide. Brains of mice sacrificed at experimental time points were harvested for immunohistochemistry and stem cell culture. Following enrichment of tumour cells from cultured brains, in vitro stem cell assays and flow characterization were performed. RESULTS: Initial experiments revealed that drug-treated cells showed an increase in self-renewal post-treatment, and an enrichment of CD15+ and CD133+ BTICs by flow analysis. To further study the response of MB cells to therapy in their orthotropic environment, in vivo experiments were performed. Through flow analysis, we found that MB cells harvested from tumor bearing mice treated with radiation and chemotherapy were enriched for stem cell markers CD133 and Sox2. The treated cells also displayed an enhanced self-renewal capacity. These studies confirm the presence of a treatment-refractory population with stem cell properties. CONCLUSIONS: Comparative BTIC profiles of surviving and relapsed mice will identify BTICs that respond to or evade specific therapy. Profiling genomic changes in “treatment-responsive” tumors against those that fail therapy will generate a differential profile of the refractory BTIC, which may guide future therapeutic approaches targeting this cell, and will serve as a model for targeting such CSCs in other TIC-driven solid tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0101. RAF INHIBITOR RESISTANCE AND PARADOXICAL ACTIVATION OF KIAA1549-BRAF FUSIONS FOUND IN PEDIATRIC LOW-GRADE ASTROCYTOMAS

Angela Sievert 1, Shih-Shan Lang 1, Katie Boucher 1, Peter Madsen 1, Erin Slaunwhite 1, Namrata Choudhari 1, Meghan Kellet 1, Phillip Storm 1, Adam Resnick 1

Abstract

INTRODUCTION: Astrocytomas are the most common type of brain tumor found in children. Activated BRAF mutations are unifying feature of this heterogeneous group of tumors with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and V600EBRAF alterations characterizing higher-grade tumors. BRAF targeted therapies such as vemurafenib have proven oncogenic BRAF signaling can be inhibited in V600E-dependent melanomas. Like the canonical V600EBRAF mutant, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation. However, here we characterize the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. METHOD: There are no established pediatric low-grade astrocytoma cell lines that harbor the BRAF fusion; therefore, we generated stably expressing BRAF fusion cell lines in Ba/F3, NIH/3T3, and murine neurosphere cells. Mutant BRAF constructs were subcloned into a Gateway compatible pMXs-Puro Retroviral Vector. Cells were infected with retrovirus and selected for stable expression with puromycin. Selective BRAF inhibition studies were performed in the presence of increasing concentrations of PLX4720, a first generation BRAF-specific inhibitor or PLX PB-3, a second generation “paradox-breaker” inhibitor (Plexxikon Inc). BRAF dimerization and KSR interaction studies were performed following transfections of HEK293Ts or NIH/3T3s. RESULTS: We find that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrate KIAA1549-BRAF fusion mediated signaling is diminished with the disruption of the BRAF kinase dimer interface. Additionally, the KIAA1549-BRAF fusion displays increased binding affinity to KSR, a RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. CONCLUSIONS: BRAF fusions, typically found in low-grade astrocytomas, are resistant to selective first-generation BRAF inhibitors designed to target V600EBRAF mutant cancers. Instead, first-generation BRAF inhibitors can induce paradoxical activation resulting in increased tumor growth in cells harboring BRAF fusions. We believe this is due to key cell signaling characteristics associated with BRAF fusions versus V600EBRAF. Second-generation selective BRAF inhibitors offer promise that direct targeting of BRAF fusions is still possible. Our data support an approach in which pediatric astrocytoma therapies must be tailored to the specific mutational context and the distinct mechanisms of action of the mutant kinase.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0102. TARGETING DNA DAMAGE RESPONSE PATHWAYS TO OVERCOME ALKYLATING AGENT RESISTANCE IN PEDIATRIC GLIOBLASTOMA

Sameer Agnihotri 1, Kelly Burrell 1, Nestor Fernandez 1, Brian Golbourn 1, Ian Clarke 1, Mark Barszczyk 1, Nesrin Sabha 1, Peter Dirks 1, Chris Jones 3, James Rutka 1, Gelareh Zadeh 1, Cynthia Hawkins 2

Abstract

INTRODUCTION: Pediatric glioblastoma (pGBM) is among the most lethal primary brain tumors in children. Current therapy includes a combination of surgery, radiation and chemotherapy, typically an alkylating agent temozolamide (TMZ). However, TMZ resistance is common and in many cases cannot be attributed to O-6-methylguanine-DNA methyltransferase (MGMT) expression. We hypothesized that other DNA damage response (DDR) pathways may account for the treatment resistance seen in pGBM. Here we report that the DNA repair proteins N-methylpurine-DNA glycosylase (MPG), involved in base excision repair (BER), and Ataxia telangiectasia mutated (ATM), a master regulator of the DDR pathway, contribute towards alkylating agent resistance in pGBM. METHOD: Primary pGBM cultures were grown in neural basal media with EGF, FGF and B27 growth factors. pGBM cell lines (SJG2 and KNS42) were maintained in DMEM-F12 and 10% FBS. Stable knockdowns of MPG, ATM or both were achieved by retroviral transduction of MPG or ATM specific shRNA constructs. Cell readouts were performed using apoptosis assays (Cleaved Caspase 3/7), cell viability (trypan blue exclusion, cell count), and DNA damage (Comet Tail assay) in the presence or absence of temozolomide/BCNU. In vivo work and MRI imaging of mice harbouring SJG2 pGBM xenografts treated with vehicle or temozolomide were done using standard procedures. RESULTS: Silencing MPG expression in TMZ-resistant pGBM cell lines and primary cultures enhanced TMZ responsiveness, while exogenously expressing MPG in TMZ-sensitive lines conferred resistance. Surprisingly, we identified a novel MPG phosphorylation site regulated by ATM kinase. Loss of phospho-MPG (serine 172) reduced MPG's ability to protect cancer cells against temozolomide and other alkylating agents. Dual targeting of ATM and MPG led to a synergistic increase in survival using an in vivo orthotopic model of pGBM treated with TMZ. Moreover, we identified a small molecule inhibitor of base excision repair, methoxyamine, as a potent sensitizer to TMZ in vitro and in vivo. CONCLUSIONS: Targeting of MPG through inhibition of BER may lead to promising new therapeutic strategies for the treatment of pGBM. In support of this, high nuclear MPG expression was observed in pGBM (60/80 samples) and not surrounding normal brain. Nuclear MPG also correlated with poorer overall survival in pGBM in one of our datasets. Evidence to date suggests that MGMT, another key determinant of TMZ resistance cannot accurately predict overall survival or response to TMZ in pGBM. We hypothesize that the BER pathway, mediated through an ATM-MPG axis, may be a key promoter of alkylating agent resistance in pGBM.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0103. SILENCING OF THE miR-17 ∼ 92 CLUSTER FAMILY BY SEED-TARGETING 8-mer antimiRs INHIBITS MEDULLOBLASTOMA PROGRESSION IN THE BRAIN

Brian Murphy 1, Susanna Obad 2, Laure Bihannic 1, Olivier Ayrault 1, Frederique Zindy 1, Sakari Kauppinen 3, Martine Roussel 1

Abstract

INTRODUCTION: Medulloblastoma (MB), originating in the cerebellum, is the most common malignant brain tumor in children. MB consists of four major groups; constitutive activation of the Sonic Hedgehog (SHH) signaling pathway is a hallmark of one group. Mouse and human SHH MBs exhibit increased expression of microRNAs (miRNAs) encoded by the miR-17 ∼ 92 and miR-106b ∼ 25 clusters compared to granule progenitors or post-mitotic granule neurons. Here, we assessed the therapeutic potential of 8-mer seed-targeting LNA-antimiR oligonucleotides, termed tiny LNAs, that inhibit miRNA seed families expressed by the miR-17 ∼ 92 and miR-106b ∼ 25 clusters in a mouse model of SHH MB. METHOD: MiR-17 ∼ 92 levels were assessed in spontaneously arisen MBs from [Ptch1 + /-;Cdkn2c-/-] and [Ptch1 + /-;Trp53-/-] mice. MBs were cultured and treated in vitro with tiny LNAs. The percentage of cells incorporating FAM-labeled tiny LNAs, Ki-67 immunoreactivity and BrdU incorporation were determined after tiny LNA treatment. Pre-treated cells were transplanted into the flanks of CD-1 nude mice to test the effect of tiny LNAs on tumor growth. Lastly, tiny LNA naïve tumor cells were transplanted into the flanks and cortices of recipient mice and treated intravenously with saline formulated tiny LNAs to assess whether systemic delivery of tiny LNAs could suppress MB growth. RESULTS: We found miR-19a and miR-17/miR-20a to be the highest expressed miRNAs from the miR-17 ∼ 92 cluster within MBs from [Ptch1 + /-;Cdkn2c-/-] and [Ptch1 + /-;Trp53-/-] mice. When treated in culture with tiny LNAs, SHH tumor cells passively and effectively took up tiny LNAs, and specifically inhibited targeted miRNA seed-sharing family members. Inhibition of miRs-17/20a/106b/93 and miR-19a/19b-1 by antimiR-17 and antimiR-19, respectively, resulted in diminished tumor cell proliferation in vitro. When tiny LNA pre-treated tumor cells were transplanted into the flanks of recipient mice, tumor growth was suppressed. Importantly, systemic delivery of tiny LNAs into flank or brain allograft-bearing mice suppressed SHH-subgroup MB progression. CONCLUSIONS: We investigated the role of miR-17/20a/106b/miR-93 and miR-19a/b in MB proliferation by inhibiting their function using 8-mer LNA-modified antimiRs directed against their seed sequences, designated as antimiR-17 and antimiR-19. Results from this study imply that 8-mer LNA-antimiRs targeting the miR-17 ∼ 92 cluster have therapeutic potential for treatment of human SHH MBs; thus, warranting consideration in the treatment of these devastating cancers. This will not only significantly advance the field of MB research, but also the cancer field as a whole by demonstrating the utility of systemically delivered tiny LNAs for the treatment of brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0104. A RETROVIRAL GENE-TRAP SCREEN IDENTIFIES PTEN INDUCED KINASE1 (PINK1) AS A NEGATIVE REGULATOR OF AEROBIC GLYCOLYSIS IN PEDIATRIC AND ADULT GLIOBLASTOMA

Brian Golbourn 1, Sameer Agnihotri 1, Rob Cairns 2, Paul Mischel 3, Kenneth Aldape 4, Cynthia Hawkins 1, Gelareh Zadeh 1, James Rutka 1

Abstract

INTRODUCTION: Pediatric glioblastoma (pGBM) comprises of 8-12% of primary CNS tumours in children and adult Glioblastoma (GBM) is the most common and lethal of all gliomas. Mutual to both is tumor aggressiveness and poor survival despite surgery, radiation and chemotherapy. Using a retroviral gene-trap screen on astrocytes cultures isolated from newborn transgenic mice predisposed to developing glioma we identified several novel genes not previously implicated in GBM and pGBM. Of great interest was PTEN induced Kinase 1, PINK1, a mitochondrial serine/threonine kinase that we demonstrate to be lost in GBM and pGBM and funtions to oppose tumour metabolism. METHOD: Astrocyte cultures isolated from RasB8 mice, which harbour activated Ras and spontaneously develop gliomas were infected with a retroviral gene-trap cassette (multiplicity of infection ∼1). Growth in soft agar was used as a measure of transformation. Isolated clones were sequenced using inverse PCR to identify insertion sites. Functional validation of PINK1 in human GBM and PHGG cells lines was accomplished by generating pooled stable over expressers of PINK1 or pooled stable knockdowns of PINK1 in fetal human astrocytes. Functional readouts were proliferation (BrdU assay), apoptosis (Caspase 3/7), metabolism (O2 consumption, lactate and glucose uptake) and reactive oxygen species measurements (ROS). RESULTS: Loss of PINK1 was observed in pediatric and adult glioma lines. PINK1 decreased expression was observed in 65% of GBM and correlated with poorer overall survival. Current work is investigating the prognostic value of PINK1 expression loss in pGBM. Functionally, PINK1 loss in fetal normal human astrocytes resulted in increased reactive oxygen species (ROS) leading to increased proliferation. In addition PINK1 deficient cells underwent metabolic remodelling and acquired a preferential aerobic glycolysis state in which O2 consumption decreased, lactate production and glucose uptake increased. Re-expression of PINK1 in pediatric and adult glioma lines reduced proliferation, ROS and disrupted tumour metabolism. CONCLUSIONS: Gene-trapping strategies in robust animal models provide an invaluable tool that complement large scale cancer genome sequencing projects in identification of relevant driver GBM modifier genes in a random non-biased manner. Furthermore, this is the first report linking PINK1 a gene involved in Parkinson's disease as a key effector of tumour metabolism. Future work is investigating loss of PINK1 in transgenic models of adult and pediatric glioma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0106. PEDIATRIC GANGLIOGLIOMAS INCLUDING THOSE IN THE BRAINSTEM SHOW BRAF V600E MUTATION IN A HIGH PERCENTAGE OF CASES

Sarah Rush 1, Andrew Donson 2, Bette Kleinschmidt-DeMasters 2, Lynne Bemis 2, Diane Birks 2, Michael Chan 2, Amy Smith 3, Michael Handler 2, Nicholas Foreman 2

Abstract

INTRODUCTION: Standard treatment for ganglioglioma (GG) currently consists of optimal tumor resection, which is curative in the majority of cases. Whether due to surgical unresectability or differing tumor biology, those GG arising in the brainstem have a poor prognosis. Effective treatment for these tumors has not been established. Therapeutically targetable mutations in the BRAF gene have recently been identified in GGs, but these cohorts contained few brainstem cases. METHOD: The present study screened BRAF mutations in a relatively large series of brainstem GGs (n = 12) and one brainstem gangliocytoma; also including 10 non-brainstem GGs and 9 brainstem pilocytic astrocytomas (PA) for comparison. All samples were screened for BRAF exon 15 mutations, the exon that encompasses the common BRAF V600E mutation. Additionally, the influence of BRAF V600E on GG biology was further evaluated by HG-U133plus2 gene expression microarray analysis of GG that carried BRAF V600E (n = 5) versus those that did not (n = 3). RESULTS: BRAF V600E was identified in 8 of 12 (67%) brainstem GG, 5 of 10 (50%) non-brainstem GGs and 1 of 8 (13%) brainstem PA. A V600-W604 deletion was observed in the gangliocytoma and one non-brainstem GG harbored H585Y. Further screening identified no mutations in any of the 18 BRAF exons in 2 of the 4 wild-type BRAF exon 15 brainstem GG that had evaluable material. Microarray analysis identified a number of genes that were significantly associated with BRAF V600E in GG, most notably IMP3 (IGF2BP3), which has previously been identified by our laboratory as an adverse prognostic factor in PA. CONCLUSIONS: Given the high percentage of pediatric brainstem GGs with BRAF V600E mutations, further studies utilizing targeted BRAF inhibitors, such as vemurafinib, may be warranted for this poor prognosis subset of GGs. Further analysis of the relationship between IMP3 and BRAF mutations also merits future studies as these both have prognostic implications and may alter therapy selections.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0107. IN VIVO AND FUNCTIONAL ANALYSIS OF NOVEL MAPK PATHWAY ACTIVATING MUTATIONS IN CHILDHOOD ASTROCYTOMA

Jan Gronych 1, David TW Jones 1, Marc Zuckermann 1, Sonja Hutter 1, Andrey Korshunov 2, Marcel Kool 1, Marina Ryzhova 3, Guido Reifenberger 4, Stefan M Pfister 1, Peter Lichter 1

Abstract

INTRODUCTION: Pilocytic astrocytoma (PA), the most common childhood brain tumor, can occur throughout the central nervous system. These tumors harbor oncogenic alterations of the MAPK/ERK pathway. While most of the cerebellar tumors mainly show alterations of BRAF, the majority of cases without identified MAPK alterations arise outside the cerebellum. Although survival rates generally are excellent, these cases often are not amenable to complete surgical resection resulting in multiple recurrences and a considerably reduced quality of life. Using deep sequencing techniques we have identified alternative MAPK alterations in these tumors, which are now subject to functional investigation in vitro and in vivo. METHOD: To investigate the downstream signaling of the identified mutations on a protein level, we have assessed their potential to activate MAPK signaling and possible synergistic combinations of alterations using in vitro models. Furthermore, since a new BRAF mutation we have identified suggests a novel machanism of enhanced dimerization of the mutant leading to constitutive activation, we looked at protein-protein binding using co-immunoprecipitation. For in vivo analysis, the mutant genes will be delivered to nestin-positive cells of the neonatal mouse cerebellum using the RCAS/Tv-a technique testing them for their tumorigenic potential. RESULTS: We have identified point mutations which have not been described in PAs so far. The most striking alterations were two hot spot mutations of the FGFR1 tyrosine kinase with co-occuring PTPN11 mutations in some cases. In vitro, the FGFR1 mutations induced MAPK signaling while expression of mutant PTPN11 alone did not induce Erk-phosphorylation. In combination with mutated FGFR1, however, it further increased MAPK activation in a synergistic manner. Additionally, we identified a novel insertion mutation of the BRAF oncogene (InsVLR). Structural data suggested that this could confer constitutive activity through enhanced dimerization which we verified by co-immunoprecipitation. CONCLUSIONS: We have functionally characterized novel mutations leading to constitutive MAPK activation in PAs. To ultimately prove their causal role in PA pathogenesis, these mutations are currently being tested for their oncogenic potential in animal models using retroviral gene transfer in neonatal mice (RCAS/Tv-a) - similar to our previously reported BRAF V600E model for PA. In the case of successful tumor induction, the established animal model will be utilized for pre-clinical testing of targeted RTK/MAPK inhibitors to eventually improve treatment of PAs harboring the respective mutations.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0108. THE METHYLOMIC LANDSCAPE OF CHILDHOOD MEDULLOBLASTOMA

David T W Jones 1, Volker Hovestadt 2, Simone Picelli 2, Wei Wang 2, Paul A Northcott 1, Marcel Kool 1, Natalie Jäger 3, Guido Reifenberger 4, Stefan Rutkowski 5, Torsten Pietsch 6, Marc Sultan 7, Marie-Laure Yaspo 7, Pablo Landgraf 8, Roland Eils 3, Andrey Korshunov 9, Marc Zapatka 2, Stefan M Pfister 1, Bernhard Radlwimmer 2, Peter Lichter 2; on behalf of the ICGC PedBrain Tumor Project10

Abstract

INTRODUCTION: Much has recently been discovered with respect to genomic and transcriptomic alterations underlying medulloblastoma, the most common embryonal brain tumor. One of the most important insights from recent studies is that medulloblastoma is not a single disease, but rather comprises four core molecular subgroups. Whilst the WNT and SHH subgroups are relatively well characterized in terms of key pathways and alterations involved in tumorigenesis, Group 3 and Group 4 tumors remain poorly understood despite extensive genomic screens. We therefore sought to characterize global epigenetic alterations occurring in medulloblastoma as part of the International Cancer Genome Project (ICGC) PedBrain Tumor project. METHOD: In order to get a global, base-resolution profile of the medulloblastoma methylome, we performed high-coverage whole-genome bisulfite sequencing on a total of 34 primary tumors and 8 normal cerebellar samples. In-house algorithms were designed in order to optimize mapping and downstream analysis of bisulfite-converted DNA reads. To supplement this, we also conducted genome-wide methylome analysis on over 300 primary medulloblastomas (frozen and FFPE) using the Illumina Infinium HumanMethylation450 bead array. Matched transcriptome data, either from Affymetrix U133 Plus2.0 arrays or RNA sequencing, as well as miRNA sequencing data, was available for over 100 tumors, allowing correlation of methylation with expression. RESULTS: Unsupervised clustering of variably methylated CpGs was highly concordant with known gene expression subgroups, indicating clear epigenetic differences in these core groups. DNA methylation correlated with overall expression levels and alternative isoforms in numerous regions. Interestingly, the strongest association was not at classical CpG islands, but downstream of transcription start sites in areas of low CpG density. Differential methylation/expression between subgroups was observed for many known subgroup markers, but also putative novel medulloblastoma oncogenes and miRNAs. Large-scale partially methylated domains (PMDs), identified in WNT and Group 3 tumors, were correlated with low gene expression and an increased somatic mutation rate. CONCLUSIONS: This study provides an extremely detailed view of the methylomic landscape of medulloblastoma, revealing novel insights into the epigenetic regulation of subgroup-specific mRNA and miRNA expression for both known and novel candidates. Furthermore, the scope of the study has allowed us to identify associations between DNA methylation, gene expression, and alternative splicing / promoter usage which have wider implications for basic biology. These data represent a significant advance in our understanding of the biological heterogeneity in medulloblastoma, which will provide a basis both for future research studies and for the development of enhanced therapeutic modalities.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0109. MicroRNA SIGNATURE OF PEDIATRIC GLIOBLASTOMA INVASION: AN IN VIVO STUDY IN PATIENT-TUMOR DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS

Yulun Huang 2, Hua Mao 1, Yue Wang 1, Mari Kogiso 1, Xiumei Zhao 1, Patricia Baxter 1, Chris Man 1, Zhong Wang 2, Youxing Zhou 2, Xiao-Nan Li 1

Abstract

INTRODUCTION: Diffuse invasion into normal brain tissue is one of the most important biologic features that make GBM particularly difficult to treat. Efforts of developing new targeted therapies against GBM invasion are hampered by our poor understanding of the invasion process, primarily because the invasive GBM cells are rarely available for comparative analysis. MicroRNAs have been implicated in regulating diverse cellular pathways; their role in mediating glioma invasion has not been fully explored. We hypothesize that direct comparison of the matched invasive and tumor core GBM cells would facilitate the discovery of key genetic changes driving GBM invasion. METHOD: To isolate matched pairs of invasive and tumor core GBM cells, whole mouse brains from a panel of 7(1 adult and 6 pediatric) patient-tumor derived orthotopic xenograft mouse models were cut into 1 mm slices, followed by microscopic dissection of tumor mass from the "normal" mouse brains from where the invasive GBM cells were isolated. MicroRNA expression was profiled with Taqman MicroRNA array (768 microRNAs); and whole genome gene expression with Affymetrics chips. To identify the affected target genes, we correlated the mRNA levels of the predicted microRNA targets with the changes of the dysregulated microRNAs. RESULTS: We identified 23 microRNAs that were upregulated and 22 microRNAs decreased in the invasive GBM cells in at least 5 of the 7 GBM models. While some of the microRNAs have been previously associated with tumor invasion (such as miR-126 and miR-138) or deyregulated in multiple human tumors (such as miR-18a and miR-383), most of the identified microRNAs have not been associated with GBM invasion. When the mRNA expression levels of the predicted genes were examined, the microRNAs with elevated expression in the invasive front appear to have better reverse correlation than those microRNAs that were down regulated. CONCLUSIONS: In conclusion, we have established a novel in vivo model system that allows for isolation of paired and biologically accurate invasive and tumor core GBM cells in vivo in mouse brains. Using this system, we have identified a novel subset of microRNAs that are significantly altered in the invasive cells. This group of microRNAs warrants further evaluation as potential therapeutic targets.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0110. ABC TRANSPORTERS, ABCG2 AND MDR, LIMIT THE EFFICACY OF DASATINIB IN A DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) MOUSE MODEL.

Alexander H Chung 1, Donna Crabtree 1, Kristin Schroeder 1, Oren J Becher 1

Abstract

INTRODUCTION: DIPG is an incurable tumor that arises in the brainstem of children. Our lab evaluates novel inhibitors in a DIPG mouse model to help prioritize the translation of novel agents. The DIPG mouse model is generated by expressing PDGF-B and Cre in nestin progenitors of the neonatal brainstem of nestin tv-a; p53 floxed mice. Dasatinib, an inhibitor of PDGFR-A and SRC activation, is currently being evaluated in clinical trials for children with DIPG. We investigated whether dasatinib, an MDR and ABCG2 substrate, would have significantly higher anti-tumor activity in a DIPG mouse model deficient for ABCG2 and MDRa. METHOD: We infected the brainstem of neonatal nestin tv-a; floxed p53; abcb1a−/-; abcg2−/- mice (ABC KO) and nestin tv-a; floxed p53; abcb1a+/+; abcg2+/+ mice (ABC WT) with PDGF-B and Cre at postnatal D4-5. At the first sign of a tumor, mice were treated with one dose of dasatinib or vehicle (25 mg/kg) by IP injection. We treated at least 4 mice per group (ABC KO-drug, ABC KO-vehicle, ABC WT-drug, ABC WT-vehicle). Treated mice were sacrificed 24 hours later and brains were extracted and placed in formalin. FFPE sections were stained for cleaved caspase-3(CC3) and phospho-histone H3 (pH3) by immunohistochemistry. RESULTS: There was no significant survival difference between DIPG-bearing mice in ABC KO and ABC WT mice. One dose of dasatinib to DIPG-bearing ABC KO mice resulted in a significant increase in CC3 levels relative to vehicle (p = 0.03) while one dose of dasatinib to DIPG-bearing ABC WT mice did not result in a significant increase in CC3 levels relative to vehicle (p = 0.30). Treatment with dasatinib resulted in 20-fold higher levels of CC3 levels in DIPGs of ABC KO mice relative to DIPGs of ABC WT mice (p = 0.002). pH3 levels did not change significantly in response to dasatinib in both models. CONCLUSIONS: ABC transporter proteins play an important role in limiting the efficacy of dasatinib in treating a PDGF-driven DIPG mouse model. A survival study is ongoing to determine if dasatinib can significantly prolong survival in a cohort of DIPG-bearing ABC KO mouse. We are also evaluating if elacridar (ABCG2, MDR inhibitor) can enhance the efficacy of dasatinib in DIPG-bearing ABC WT mice.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0111. IN VIVO CYTOTOXICITY OF TEMOZOLOMIDE AGAINST DAOY CELLS MAY BE POTENTIATED BY ENZYMATIC DEPLETION OF ASPARAGINE/GLUTAMINE

Eduard Panosyan 1, Yuntao Wang 1, Joseph Lasky 1

Abstract

INTRODUCTION: Enzymatic depletion of amino acids for therapeutic purposes is understudied in brain tumors. Asparaginase (ASNase) effectively contributes in acute leukemia eradication by decreasing asparagine levels in serum and CSF without crossing the blood-brain barrier. Cancer cells may become ASNase resistant by up-regulation of asparagine synthetase (ASNS), higher expression of which has been linked to biological aggressiveness of various tumors, and perhaps including glial malignancies [probesetanalyzer.com]. We tested if depletion of asparagine and glutamine may augment brain tumor cell death in vitro or in vivo. METHOD: First, in vitro enzymatic treatment with ASNase (or inhibition of glutamine synthetase by methionine sulfoximine) demonstrated activity against brain tumor cell lines (MTS assay), which can be reversed by glutamine supplementation. ASNS expression (by PCR) is likely to be responsible for variable sensitivity patterns observed in our cell lines. Given that our initial in vitro data suggested possible synergistic effect of ASNase and cytotoxic agents, we have utilized heterotopic subcutaneous DAOY xenografts (1x10e7 cells/animal) in SCID mice to test Temozolomide and ASNase combination. Over three weeks, transplanted animals (n = 5 in each group) were treated with IP ASNase and/or temozolomide. RESULTS: Temozolomide dose was (2 IU/g/dose M/W/F x 2 weeks) and asparaginase dose was (0.05mg/g/dose x 5 days/week x 2 weeks). Tumor growth kinetics were compared and included untreated controls (serum asparagine depletion in ASNase treated animals was confirmed with HPLC previously). In Vivo data showed no effect with ASNase mono-therapy; however significant growth suppression was observed by ASNase and temozolomide combination compared with modest growth suppression by temozolomide only (tumors were twice smaller with combined treatment, p = 0.036). This effect started in 1 week after initiation of therapy and lasted until 3 weeks, when curves started to remerge. CONCLUSIONS: We concluded that pharmacological depletion of selected amino acids (asparagine and glutamine) may potentially serve as an adjunct to enhance effect of cytotoxic therapy against brain tumors. We plan to use intracranial models in next experiments with parallel pharmacodynamic measurements to further elucidate potential role of amino acid depletion in brain tumor therapeutics.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0112. A PATIENT TUMOR-DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODEL REPLICATING THE GROUP 3 SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMOR IN CHILDREN

Zhigang Liu 1, Xiumei Zhao 2, Yue Wang 2, Hua Mao 2, Yulun Huang 2, Mari Kogiso 2, Patricia Baxter 2, Adekunle Adesina 2, Jack Su 2, Daniel Picard 3, Annie Huang 3, Laszlo Perlaky 2, Murali Chintagumpala 2, Ching Lau 2, Susan Blaney 2, Xiao-Nan Li 2

Abstract

INTRODUCTION: Supratentorial primitive neuroectodermal tumor (sPNET) is a highly malignant brain tumor with poor prognosis. New models that replicate the molecular subtypes and maintains the cancer stem cells (CSCs) of this deadly disease is highly desired. Here, we describe the establishment of a novel transplantable orthotopic xenograft mouse model through direct injection of fresh surgical specimen of a pediatric sPNET into the right cerebrum of Rag2/SCID mice. METHOD: Fresh tumor specimen from a 14-year-old female was obtained in the cryostat laboratory following an IRB approved protocol and implanted directly into the right cerebral sphere of Rag2/SCID mice. Serial subtransplantation was performed by harvesting the developed xenograft tumors and injecting into the brains of recipient animals. Xenograft tumors were characterized through histopathological analysis, and subclassified using the newly discovered molecular makers of group 1, 2 and 3. The presence of cancer stem cell markers (CD133, CD15, CD24, CD44 and CD117) was examined with flow cytometry. A permanent neurosphere line was established through incubation of xenograft cells in serum-free medium supplemented with EGF and bFGF. RESULTS: The xenograft tumors (IC-2664PNT) replicated the histopathological features of the original patient tumor and have been serially subtransplanted for more than 5 times. Quantitative RT-PCR analysis identified TWIST1 and FOXJ1 as the over-expressed genes, thereby categorizing this model into Group 3. Flow cytometry analysis of putative cancer stem cell markers in xenograft tumors during serial sub-transplantation identified CD133+ and CD15+ cells as the major subpopulations. Functionally, dual positive (D133+/CD15+) cells were found to possess the highest neurosphere forming efficiency in vitro and the strongest tumor forming capacity in vivo in mouse brains. A matching neurosphere line BXD-2664PNT was established. CONCLUSIONS: We have successfully established a patient tumor-derived orthotopic xenograft mouse model that replicated the biology of group 3 childhood sPNET as well as a matching neurosphere line. Using this unique model system, we isolated and characterized CSCs for childhood sPNET. We confirmed the presence of mixed subpopulations of sPNET cells with strong self-renewal and tumor initiating capacities, and identified CD133 and CD15 (mono- or dual-positive) as major surface antigen markers of sPNET CSCs. This novel model system has thus provided us with a unique opportunity to investigate the biology and test new therapies against group 3 sPNET tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0113. HUMANIZED MOUSE MODELS FOR MEDULLOBLASTOMA

Miller Huang 1, Anders Persson 1, Fredrik Swartling 5, Branden Moriarity 3, Faith Kreitzer 2, David Largaespada 3, Bruce Conklin 2, Michael Taylor 4, William Weiss 1

Abstract

INTRODUCTION: Medulloblastoma is a common and frequently lethal tumor of childhood. A third of patients either fail to respond or relapse after an initial response, while disabling cognitive defects are observed in a majority of long term survivors. Our ability to treat these patients and spare normal growth cognition depends on understanding key drivers and identifying therapeutic targets in this disease. Genome wide sequencing efforts have revealed a paucity of driver mutations. In contrast, somatic copy number variations (SCNV) are common, robust, and show specificity within subgroups. However, modeling SCNVs is challenging, as human and mouse chromosomes do not align completely. METHOD: We are generating iPS-cell based in vivo models of medulloblastoma, which enable us to model SCNVs. First, we are differentiating human iPSC toward two distinct and presumed cells of origin of medulloblastoma: neural stem cells (NSC) and granule neural precursor cells (GNPC). Separately, we are developing Transcription Activator-Like Effector Nucleases (TALEN) to knockout TP53 and PTCH1 to predispose these cells toward a medulloblastoma fate. Previously, we found that stabilized MYCN (MYCNT58A) can drive medulloblastoma in mouse cells. Here, we are transducing MYCNT58A into human NSC and GNPC, followed by orthotopic transplantation into hindbrains of immunocompromised mice. RESULTS: Testing multiple differentiation protocols, we have successfully generated NSCs from our human iPSCs, as evidenced by positive staining for NSC markers Nestin, Sox1, Sox2 and Pax6, and negative staining for the pluripotency marker Oct4. In addition, we have successfully generated GNPC derived from human iPSCs, evidenced by positive staining for GNPC markers Math1, En1, Gbx2 and HoxA2. We have also developed active TALENs against TP53 and PTCH1. These TALENs were introduced into human cells, and exhibit nuclease activity in a Surveyor assay, a measure of specific cleavage of genomic DNA at TP53 and PTCH1. CONCLUSIONS: We have successfully differentiated iPS cells towards NSC and GNPC and developed active TALENs targeting TP53 and PTCH1. In parallel, we are developing chromosomal engineering methodology to model SCNV. The resulting models promise to clarify both the biology and genetics of this disease, and can be integrated into treatment algorithms for children with high-risk subgroups of medulloblastoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0114. MEDULLOBLASTOMA METASTASES DO NOT ARISE FROM THE TUMOR INITIATING CELL COMPARTMENT OF THE PRIMARY TUMOR

Livia Garzia 1, Sorana Morrissy 1, Kory Zayne 1, Xiaochong Wu 1, Peter Dirks 1, John Dick 2, Lincoln Stein 3, Lara Collier 6, David Largaespada 4, Adam Dupuy 5, Michael Taylor 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common paediatric malignant brain tumor. By the way of optimal surgery, radiation, and chemotherapy, medulloblastoma can be treated in a good fraction of children, but despite the best therapy the outcome for patients with metastases is still poor. Accordingly to the cancer stem cell (CSC) theory only a fraction of the tumor (Tumor Initiating Cells – TICs) are able to transplant the disease in immunocompromised recipients, thereby recapitulating the original tumor. We hypothesized there that TICs in the primary tumor are the best candidates to disseminate through the CSF and give rise to distant metastases. METHOD: We developed a novel murine model of metastatic MB, which is highly penetrant, has a short latency, and involves random secondary genetic events. The model is based on mobilizing the Sleeping Beauty transposon (SB) in the cerebella of Ptch+/- mice. Individual insertions of SB serve as highly specific lineage markers and can be used with high confidence to determine hierarchical relationships. We flow-sorted for the known medulloblastoma TIC marker: the CD15 antigen, to compare the TIC-enriched and TIC-depleted fractions, then sequenced millions of transposon insertion sequences (Illumina) from the two fractions, as well as the matching leptomeningeal metastases. RESULTS: TIC enriched cells from SB medulloblastoma are more efficient in transplanting MB to immunocompromised recipient animals. However, several statistical approaches used to compare the TIC-enriched and TIC-depleted fractions to the leptomeningeal metastases from the same animal(s), revealed that metastases are very different from both the TIC-enriched and the TIC-depleted fractions. Furthermore the two compartments are equally probable to give rise to MB metastasis, suggesting that metastases are unlikely to arise predominantly from the primary tumor TIC compartment. CONCLUSIONS: MB metastases are genetically distinct from both the TIC enriched and TIC depleted fractions. Our data suggest that the metastatic TIC (mTIC) is distinct from the primary tumor TIC (pTIC), that eradication of the pTIC compartment might not impact on metastatic disease, and that identification of the mTIC could help to design more effective therapies for patients with metastatic medulloblastoma

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0115. INVESTIGATION OF MUTATIONS IN EXON 4 OF IDH1 AND IDH2 GENES IN LOW GRADE ASTROCYTOMAS OF CHILDHOOD

Gabriela Rampazzo 1, Lais Moraes 2, Mario Paniago 1, Indhira Oliveira 1, Nasjla Silva 1, Andrea Cappellano 1, Sergio Cavalheiro 1, Maria Teresa Alves 3, Janete Cerutti 2, Silvia Toledo 1

Abstract

INTRODUCTION: About 30% of tumors of the central nervous system in children are low-grade astrocytomas. Until recently, little was known about the molecular background of pediatric low-grade gliomas. Alteration of cellular metabolism has been proposed as a novel oncogenetic mechanism. The genes encoding isocitrate dehydrogenases IDH1 and IDH2 – cytoplasmic and mitochondrial, respectively - are recurrently mutated in different cancers. Earlier studies demonstrated missense mutations in IDH genes, specific for residues R132, in IDH1; and R172 and R140 in IDH2, both in exon 4. In our study we analyze the presence of mutations in IDH1 and IDH2, in low-grade childhood astrocytomas. METHOD: Eighty-four samples of low grade astrocytomas were obtained from patients attending the IOP-GRAACC/UNIFESP (Pediatric Oncology Institute - Grupo de Apoio a Criança e ao Adolescente com Cancer/Federal University of São Paulo), including 61 grade I astrocytomas (A-I) and 23 grade II astrocytomas (A–II). Exon 4 of both genes, from each sample, were amplified by polymerase chain reactions (PCR) and sequenced to identify specific mutations. RESULTS: From our cohort, 22 patients were 0-5 and 62 were older than 5 years old at diagnosis. Additionally, 45 patients were more than five years free survival, 15 showed stable disease, 7 showed disease progression and 20 patients died of disease progression. Only two samples showed mutations in the R132/R172 residues. Between samples carring the R132/R172 mutation, one patient has A-II and the other one has A-I tumor. Also, we observed 10 patients with polymorphism G105G (reference SNP 11554137:C > T). Additionally, we observed two new mutations off the hotspots at IDH1 and IDH2 gene. CONCLUSIONS: We demonstrated that one A-II sample showed mutation at R132 (4%) and had a polymorphism G105G in IDH1. One A-I sample showed R172 mutation. Two mutations were found off the previously described hotspots, one in IDH1 and other in IDH2. Since we observed a smaller frequency of mutations in IDHs than previously described and there is growing evidence for widespread presence of these mutations in different cancers, we assume that the expected mutations may not necessarily be in exon 4, but also in other regions of the gene.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0116. INTRAVENOUS INJECTION OF ONCOLYTIC PICORNAVIRUS SVV-001 PROLONGS ANIMAL SURVIVAL IN A PANEL OF PRIMARY TUMOR-BASED ORTHOTOPIC XENOGRAFT MOUSE MODELS OF PEDIATRIC GLIOMA

Zhigang Liu 1, Xiumei Zhao 2, Hua Mao 2, Patricia Baxter 2, Yulun Huang 2, Litian Yu 2, Jack Su 2, Adesina Adekunle 2, Lazlo Perlaky 2, Mary Hurwitz 2, Richard Hurwitz 2, Ching Lau 2, Murali Chintagumpala 2, Susan Blaney 2, Xiao-Nan Li 2

Abstract

INTRODUCTION: GBM is the most malignant brain tumor that occurs both in children and in adults. Unfortunately, current treatments have proven ineffective in providing long-term survival. Research on new therapeutic means is needed. Oncolytic viruses kill cancer cells through mechanisms different from conventional therapeutics; they may not be susceptible to the same pathways of drug or radiation resistance. Seneca Valley virus (SVV-001) is a non-pathogenic oncolytic virus that can pass through the blood brain barrier. Here, we examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas in a panel of patient tumor-derived orthotopic xenograft models. METHOD: Six orthotopic xenograft mouse models (one anaplastic astrocytoma and five GBMs) were established through direct injection of fresh surgical specimens into the brains of SCID mice. In vitro anti-tumor activities were examined in primary cultures as well as in pre-formed neurospheres and in self-renewing glioma cells. In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models through a single intravenous injection of SVV-001. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids, respectively. RESULTS: SVV-001 at MOI of 0.5 to 25 replicated in and effectively killed primary cultures, pre-formed neurospheres, and self-renewing stem-like single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 x 1012 viral particle/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < 0.05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < 0.01). CONCLUSIONS: Our results demonstrated the potent anti-tumor activities of SVV-001 against pediatric gliomas. Since this study was performed in a relatively large panel of primary tumor-based orthotopic xenograft models, it provided preclinical rationale that supports the consideration of SVV-001 for clinical trials against pediatric anaplastic astrocytoma and GBM. Our results also identified the α2,3- and α2,6-linked sialic acids as key components that mediate SVV-001 infection, which represents an important first step for future identification of SVV-001 receptors that can potentially be used as diagnostic markers in pediatric GBMs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0117. EGFR ACTIVATION CONFERS RESISTANCE TO MAPK PATHWAY INHIBITORS IN GLIOMA

Jie Zhang 1, Sujatmi Hariono 1, Rintaro Hashizume 1, Qiwen Fan 1, C David James 1, William A Weiss 1, Theodore Nicolaides 1

Abstract

INTRODUCTION: Recent discoveries in cancer research have implicated activating alterations in the MAPK pathway, including BRAF-V600E missense mutation, NF1 loss, and KIAA1549:BRAF fusions, in pediatric gliomagenesis. Potent inhibitors of BRAF-V600E and MEK are being used to target this pathway in pre-clinical and clinical studies. Negative feedback loops downstream of MEK have been demonstrated in multiple tumor types, however, and may limit the efficacy of MAPK pathway inhibitors. Here we have sought to investigate the activation and consequences of this feedback loop in treating pediatric gliomas with MAPK pathway inhibitors. METHOD: Using the BRAF-V600E inhibitor PLX4720, as well as MEK inhibitors AZD-6244 and PD-0325901, we treated BRAF-V600E mutant (AM-38, DBTRG-05MG), NF1-mutant (LN229), and BRAF + NF1- wildtype glioma cell lines and their derivative xenografts, in vitro and in vivo, respectively. Treated and untreated cell lines and xenografts were compared for EGFR activation as well as other pathway signaling changes. RESULTS: Both BRAF inhibition and MEK inhibition resulted in a dose-dependent increase in EGFR phosphorylation in glioma cell lines and their derivative xenografts. This activation was associated with an increase in AKT activity and may also contribute to MAPK recovery from inhibitor treatment of BRAF-mutant gliomas. Addition of an EGFR kinase inhibitor to either BRAF or MEK inhibitor prevents EGFR/AKT activation, resulting in increased anti-tumor effect against BRAF-V600E cell lines and xenografts. CONCLUSIONS: BRAF or MEK inhibition results in EGFR activation in glioma cell lines with BRAF-V600E mutation. This appears to be a consequence of repressing a negative feedback loop downstream of MEK. Combined EGFR and MEK (or BRAF) inhibition prevents EGFR activation, resulting in improved efficacy from combination treatment, both in vitro and in vivo.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0118. NOVEL SMALL MOLECULE AND ENZYMATIC REGULATORS OF HDACS: NEW EPIGENETIC TARGETS

Peter J Madsen 1, Erin S Slaunwhite 1, Jenny F Ma 1, Rosemary E Henn 1, Adam G Hanno 1, Katie L Boucher 1, Phillip B Storm 1, Adam C Resnick 1

Abstract

INTRODUCTION: The role of epigenetics in pediatric brain tumor development has been highlighted in a number of recent studies. A more complete understanding of epigenetic mechanisms is needed to fully elucidate the pathogenesis of these tumors and potential therapeutic targets. To that end, we identify a novel regulator of class I histone deacetylases (HDAC), inositol polyphosphate multikinase (IPMK). IPMK is required for the generation of higher-order inositol polyphosphates, an under-characterized class of second messengers involved in numerous cell-signaling processes. This finding opens the door for the development of more targeted HDAC inhibitors and insights into important mechanisms of epigenetic regulation. METHOD: Protein-protein interaction screens were performed with over-expressed IPMK and HDACs 1 through 8 in HEK 293T cells with further analysis in mouse embryonic fibroblasts (MEFs) to confirm endogenous interaction. To further investigate the role of IPMK in histone acetylation, a CRE-inducible IPMK knock-out model was utilized in MEFs and the role for inositol kinase activity was confirmed with and without rescue via IPMK and kinase-dead IPMK constructs. Cell-based assays of HDAC activity were performed in this knock-out model using a reporter-based assay, followed by in vitro activity assays designed to elucidate the mechanism(s) by which IPMK regulation of HDACs occurs. RESULTS: Protein-protein interaction studies demonstrated that IPMK strongly interacts with HDAC1, HDAC2, and HDAC3. CRE-mediated loss of IPMK lead to a decrease in the acetylation of numerous histone lysine residues, while return/rescue of kinase-active IPMK restored acetylation levels. In vivo, cell-based reporter analyses of HDAC activity identified IPMK as a negative regulator of HDAC3. Additional studies elucidated the mechanism by which IPMK affects active complex formation of HDAC3 with its co-repressor SMRT. This mechanism likely depends upon the recently characterized presence of an inositol polyphosphate positioned in the interaction surface of the HDAC3/SMRT complex. CONCLUSIONS: The role of epigenetics and epigenetic dysregulation in pediatric brain tumors has been highlighted in a number of studies that identify genetic alterations in epigenetic elements. These findings emphasize the need for more research into this important and rapidly evolving area of biology. To that end, we have identified IPMK as a novel regulator of class I HDACs, particularly HDAC3. These findings provide the opportunity for new pharmacologic targets and will direct future studies into the complex regulation of HDACs. They also help to better define the diverse roles of IPKs and their enzymatic products.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0119. GENE NETWORKS IMPLICATED IN PEDIATRIC INTRACRANIAL EPENDYMOMA

Anbarasu Lourdusamy 1, Hazel Rogers 1, Jennifer Ward 1, Ruman Rahman 1, Richard Gilbertson 2, Richard Grundy 1

Abstract

INTRODUCTION: Ependymomas are the second most common malignant brain tumor in children with high mortality and heterogeneous biology that is poorly understood. In this study, we performed a systematic analysis of the intrinsic organization of pediatric ependymoma transcriptome to gain better understanding of tumor biology. METHOD: We used a systems biology approach and analysed gene expression profiles in 30 Supratentorial (ST) and 30 Posterior fossa (PF) tumour samples measured using Affymetrix hgu133-plus2 arrays. RESULTS: Network analysis identified 14 modules of varying sizes, from 36 to 1831 and six showed significant differential expression between tumour locations. The strongest tumour-location associated module (P = 5.57 × 10−19) is significantly enriched with neurogenesis and regulation of cell proliferation genes including ID2, LAMA1, LAMA2, LINGO1, LX2, MYCN, TACC2, and TNC. Interestingly, another module characterized by the up-regulation of genes in PF included multiple homeobox (HOX) family members (HOXA3, HOXA4, HOXA-AS2, HOXB2, HOXB3, HOXB-AS3, HOXC4, HOXD3 and HOXD4). Genes in 1p36 region and kinase activity are enriched in a module that include CDK3, EGFR, EPHA2, EPHB2, EPHB4, and PIK3R2. CONCLUSIONS: Our systems biology analysis of genome-wide expression data from ST and PF tumors from pediatric ependymoma (PE) patients identified dysregulated biological pathways and molecular targets underlying critical molecular alterations in PE pathogenesis. Functional studies will be used to validate novel PE genes and highlight its biological relevance.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0120. INTEGRATED ANALYSIS OF microRNA AND mRNA EXPRESSION PROFILES HIGHLIGHTS ALTERATIONS IN EPENDYMOMA

Anbarasu Lourdusamy 1, Hazel Rogers 1, Jennifer Ward 1, Ruman Rahman 1, Richard Gilbertson 2, Richard Grundy 1

Abstract

INTRODUCTION: MicroRNAs (miRNAs) are involved in tumorigenesis and tumor progression by regulating post-transcriptional gene expression. However, the miRNA-mRNA regulatory network is far from being fully understood. The objective of this study is to identify the ependymoma specific miRNAs and their target mRNAs using an integrated approach. METHOD: We performed microRNA as well as mRNA expression analysis of 64 ependymoma samples: 23 Posterior fossa (PF); 29 Supratentorial (ST); and Spinal (SP) tumors by means of microarrays. RESULTS: We identified 170 miRNAs and 1911 genes that are differentially expressed between different tumor types. Expressions of 52% miRNAs are decreased in ST compared to PF and SP. The integrated analysis of microRNA and mRNA expression pointed out regulative networks identifying HADC4, MTSS1L, SDC1, and EPHA2 as potential targets of miR-10a. Notably, a family of several miRNAs including miR-23, miR-27, and miR-30 alters the expression of EPHB2 whereas the expression of LAMA2 is altered by miR-29. Genes involved in neurogenesis (P = 1.31 × 10−29) and cytoskeletal protein binding (P = 5.83 × 10-07) are enriched in significant miRNA-mRNA anti-correlations. CONCLUSIONS: This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in ependymoma.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0121. PHASE II STUDY OF SORAFENIB IN CHILDREN WITH RECURRENT/PROGRESSIVE LOW-GRADE ASTROCYTOMAS

Matthias Karajannis 1, Michael Fisher 2, Sarah Milla 2, Kenneth Cohen 3, Genevieve Legault 1, Jeffrey Wisoff 1, David Harter 1, Amanda Merkelson 1, Michael Bloom 1, Girish Dhall 4, David Jones 5, Andrey Korshunov 5, Stefan Pfister 5, Charles Eberhart 3, Angela Sievert 2, Adam Resnick 2, David Zagzag 1, Jeffrey Allen 1

Abstract

INTRODUCTION: Activation of the RAS-RAF-MEK-ERK signaling pathway, most commonly through loss of NF1 or KIAA1549-BRAF tandem duplication, is thought to be a key driver of pediatric low-grade astrocytoma (PLGA) growth. Sorafenib is a multi-kinase inhibitor targeting BRAF, VEGFR, PDGFR and c-kit. This multi-center phase II study was performed to determine the response rate to sorafenib in children with recurrent or progressive PLGA. We are reporting clinical study results, along with complementary preclinical data using sorafenib. METHOD: Twelve patients, including three with neurofibromatosis type 1 (NF1), were enrolled on this trial. Key eligibility criteria included age ≥2 years and at least one prior standard chemotherapy treatment. Histological confirmation was required, except for optic pathway gliomas. Sorafenib was administered twice daily at 200mg/m2/dose (maximum of 400mg/dose). Magnetic resonance imaging, including three-dimensional volumetric tumor analysis, was performed after every third 28-day cycles, or earlier, if clinically indicated. Primary endpoint was radiological response. We also performed in vitro studies using sorafenib on BRAF wild-type cell lines and cell lines containing mutant BRAFV600E or BRAF-fusion constructs. RESULTS: One patient was removed from study after one week due to recurrent urosepsis. Eleven patients were therefore evaluable for response, with a mean number of prior chemotherapy regimens of 2.4 (median 2). Nine patients (82%) discontinued sorafenib after three cycles due to tumor progression, including three patients with KIAA1549-BRAF tandem duplication. One patient with a spinal cord ganglioglioma completed six cycles with stable disease. Another patient with a brainstem pilomyxoid astrocytoma achieved a partial radiological response. Results of in vitro testing indicate that sorafenib may lead to paradoxical ERK activation in both BRAF wild type and KIAA1549-BRAF mutant cell lines. CONCLUSIONS: Sorafenib appears ineffective for the treatment of recurrent or progressive PLGA, irrespective of NF1 or tumor BRAF status. We are currently performing comprehensive molecular genetic studies on the tumor tissue of the single patient who responded to treatment. The observed, unexpectedly high rate of rapid early progression in PLGA patients treated with sorafenib may indicate a potential growth-stimulating effect. In vitro testing of sorafenib suggests paradoxical ERK activation as a probable mechanism.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0122. VARIABILITY IN THE INITIAL SURGICAL TREATMENT FOR CRANIOPHARYNGIOMA: SURVEY OF THE AANS MEMBERSHIP

Todd Hankinson 1, Jacob Gump 2, Cesar Serrano-Almeida 1, Michelle Torok 2, Michael Handler 1, Arthur Liu 2, Nicholas Foreman 1

Abstract

INTRODUCTION: Craniopharyngioma is well known for the options that exist regarding initial surgical management. Recent secondary data analyses indicate that the most common traditional algorithms (gross total resection versus subtotal resection with radiation) are not employed in many cases. These data prompted us to further explore the initial practice patterns of neurosurgeons using a brief survey. We hypothesized that neurosurgeons commonly elect initial treatment strategies other than gross total surgical resection or subtotal resection followed by radiation therapy. METHOD: A nine question survey was designed and electronically distributed to 2974 AANS members as an AANS Special Announcement. Responses were collected through Zoomerang and analyzed using standard statistical techniques. RESULTS: 102 responses were collected. 36% of respondents estimated their practice includes >75% children and 61% described their practice as academic. 36%, under certain circumstances, recommend observation or radiation therapy for craniopharyngioma in the absence of tissue. 46% of these recommending this in >10% of cases. Following GTR, 90% never recommend RT. Following STR, 35% always recommend RT and 59% recommend it in >50% of cases. Following STR or biopsy alone, 18% and 11%, respectively, never recommend XRT. There were no differences in treatment patterns based on respondent demographics. Regarding observation without tissue, 66.1% in academics and 60.0% in private practice have never done so (p = 0.53). CONCLUSIONS: Survey data from a general population of neurosurgeons indicate that a considerable subset of patients with craniopharyngioma aren't treated with the most commonly described algorithms. These results are significant because they reinforce the concept that therapy for patients with craniopharyngioma is often individually tailored. This should influence clinical researchers to investigate the outcomes of patients treated using alternative methods. This will lend insight into appropriate treatment options and contribute to the design of prospective quality of life outcomes studies for patients with craniopharyngioma. Given the rarity of this tumor, pragmatic trial designs that accommodate less restrictive treatment algorithms are necessary.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0123. ONCOGENIC ROLE OF A PHOTORECEPTOR TRANSCRIPTION FACTOR, NRL, IN METASTATIC MEDULLOBLASTOMA

Alexandra Garancher 2, Nathalie Rocques 1, Catherine Miquel 3, Christian Sainte-Rose 4, Olivier Delattre 1, Franck Bourdeaut 1, Alain Eychène 1, Celio Pouponnot 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common malignant brain tumor in children. This tumor arises in the cerebellum and is highly invasive. Recent transcriptomic studies identified four MB subgroups: WNT, HH, 3 and 4. Group 3 is of poorer prognosis, frequently metastatic and refractory to current therapies. Despite these clinical features, it remains poorly characterized. It expresses genes of the "photoreceptor" a differentiation program. It is not infrequent that cancer cells express aberrant terminal differentiation programs unrelated to the tissue of origin.These aberrant differentiation programs are not thought to participate directly and actively to cancer progression. METHOD: The photoreceptor differentiation program, normally expressed in retina, has long been identified in MB but still remains an enigma. This program found in group 3 includes the photoreceptor specific transcription factor Neural Retina Leucine zipper (NRL). NRL expression is highly specific of photoreceptors in the retina, where it controls their terminal differentiation. Although it belongs to the MAF oncogene family, its oncogenic activity has not been assessed. MAF proteins have been demonstrated to be atypical oncogenes involved in terminal differentiation during normal development, but potent oncogenes in cancer. RESULTS: We are currently investigating the involvement of this key player of the photoreceptor program NRL in the MB metastatic subgroup 3.Using in vitro and in vivo approaches including in vivo orthotopic grafting, we show that overexpression of NRL in MB cell lines induces tumor growth as well as migration and invasion. This suggests that NRL can act as an oncogene in MB and could be potentially involved in the metastatic properties of subgroup 3. Our results based on shRNA mediated loss of function support a crucial role of NRL for MB progression. CONCLUSIONS: We demonstrate that NRL is required for cell cycle progression and protection from apoptosis in MB. Accordingly, NRL knock-down increases survival in orthotopic grafting experiments. We identify two NRL target genes by ChIP, the expression of which is coregulated with NRL in human MBs that are likely involved in these two activities.The current study demonstrates for the first time an oncogenic role of the photoreceptor transcription factor NRL and that the photoreceptor program participates actively in tumor progression of group 3 MB.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0124. GLIOMA-ASSOCIATED SOMATIC MUTATIONS DRIVE A RAPID CONVERSION OF NEURAL STEM CELLS INTO TUMORIGENIC OLIGODENDROGLIAL PROGENITORS

Moise Danielpour 1, Rachelle Levy 1, C Danielle Antonuk 1, Javier Rodriguez 1, Jessica Molina Aravena 1, Gi Bum Kim 1, David Gate 1, Serguei Bannykh 1, Clive Svendsen 1, Terrence Town 1, Joshua Breunig 1

Abstract

INTRODUCTION: Recent findings regarding the cell (or cells) of origin in brain tumors have yielded conflicting results. Specifically, several studies have indicated that glioma stem cells show remarkable similarity to neural stem cells (NSCs). Alternatively, a large body of evidence by multiple groups has shown that in many cases the tumor cell of origin is a rapidly proliferating oligodendrocyte progenitor cell (OPC)-like population. METHOD: We have created a novel, autochthonic, in vivo model of high grade oligodendroglioma which allows for exquisite control over the genetic determinants as well as the temporal and spatial genesis of glioma, permitting for greater insight into the cellular dynamics of tumor initiation. Somatic mutations of the RTK/Ras pathway are introduced by neonatal electroporation of plasmid DNA in combination with transposon technology. RESULTS: Within 48 hours, transgenes are expressed at a high level with coincident fluorescent protein labeling. Notably, starting at these early time points and proceeding through the first week, radial glial stem cells transform into antigenically-defined oligodendroglial-like progenitor cells, prematurely depleting the neural stem cell population. CONCLUSIONS: Thus, we conclude that naturally-occurring somatic mutations drive a rapid and virtually complete conversion of NSCs into an OPC phenotype in a manner that would be difficult to observe in other models, thus unifying the previously disparate findings.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0125. DEVELOPMENT OF A FIVE-GENE HEDGEHOG SIGNATURE AS A PATIENT PRESELECTION TOOL FOR HEDGEHOG PATHWAY-TARGETED THERAPY IN MEDULLOBLASTOMA

Dereck Amakye 1, Douglas Robinson 2, Kristine Rose 1, Yoon-Jae Cho 3, Keith L Ligon 4, Thad Sharp 1, Yuichi Ando 5, Birgit Geoerger 6, Francois Doz 7, David Ashley 8, Darren Hargrave 9, Michela Casanova 10, Hussein Tawbi 11, John Heath 12, Eric Bouffet 13, Alba A Brandes 14, Julia Chisholm 15, Jordi Rodon 16, Anne Thomas 17, Alain Mita 18, Tobey MacDonald 19, Mark Kieran 4

Abstract

INTRODUCTION: Current therapies for medulloblastoma (MB) are sub-optimal, with significant long-term toxicities and a lack of effective salvage strategies. Gene expression profiling studies have identified distinct molecular subgroups of MB, including one characterized by activated Hedgehog (Hh) signaling. In this study, bioinformatic analyses of available gene expression data were used to identify a five-gene Hh signature that can be assayed in formalin-fixed paraffin-embedded (FFPE) tumor samples by reverse transcriptase polymerase chain reaction (RT-PCR). The predictive value for tumor response of the assay was tested in adult and pediatric patients with MB enrolled in trials of the Hh inhibitor LDE225. METHOD: The five-gene signature was selected from a panel of genes associated with the Hh subgroup classification, previously determined by Affymetrix profiling in fresh frozen MB tumors. Genes shown to be differentially expressed in these tumors and amenable to RT-PCR assay development were measured in matched FFPE tumors to identify the five-gene signature. Expression levels of the signature genes were used in a predictive model to determine Hh activation status. The assay and model were validated in an independent set of MB tumors and used to evaluate tumors from patients with relapsed MB treated with LDE225 in three phase I trials. RESULTS: The five-gene signature assay for determination of Hh activation status demonstrated a perfect concordance with the status determined by Affymetrix profiling in 25 independent MB samples. Analysis of MB samples from 37 patients treated with LDE225 showed a correlation between Hh activation status and tumor response. All six patients evaluated who responded (partial or complete response by RECIST 1.0) to LDE225 treatment were predicted to have Hh pathway-activated tumors using the five-gene signature assay. Of the 31 remaining patients, 29 were predicted to have Hh pathway-nonactivated tumors and two (with stable disease) were predicted to have Hh pathway-activated tumors. CONCLUSIONS: A five-gene Hh signature assay, based on RT-PCR, was developed and validated for use as a patient preselection tool for Hh inhibitor therapy. A strong correlation between Hh activation status, as predicted by the five-gene Hh signature assay and response to LDE225 treatment was observed. The predictive value of the assay for treatment benefit in patients with recurrent MB is under further evaluation in an ongoing phase I/II trial of LDE225. In addition, the five-gene Hh assay will be used to determine eligibility in a randomized phase III trial of LDE225 versus temozolomide in patients with Hh pathway-activated relapsed MB.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0126. GLIA MATURATION FACTOR (GMFß), A MEMBER OF THE ACTIN DEPOLYMERIZATION FACTOR (ADF) SUPERFAMILY, PROMOTES GLIAL AND EMBRYONIC NEURONAL TUMOR CELL DIFFERENTIATION

David Eisenstat 1, Xiaohua Song 1

Abstract

INTRODUCTION: GMFß was first identified as a factor that promoted malignant glial cell process outgrowth in vitro. Initially there was no identified homology to other proteins or growth factors. Bioinformatics and other approaches now place this 17 kDa phosphoprotein in the ADF family with closest homology to cofilin and regulation by the MAPK pathway in vitro. GMFß is highly expressed in the central and peripheral nervous systems, with cytoplasmic expression in several cell types, including but not limited to pyramidal neurons of the cortex and Bergmann glia of the cerebellum. METHOD: We developed specific anti-peptide polyclonal antibodies to an internal motif (P01) and the C-terminus (P02) of GMFß. In addition, co-immunoprecipitation (co-IP) assays were performed with antibodies to actin. Malignant glioma cells were treated with cytochalasin D to depolymerize the actin cytoskeleton or with colchicine to disrupt microtubules. Cis-retinoic acid (RA) was used to promote neurite outgrowth and/or diiferentiation. Lambda alkaline phosphatase was used to dephosphorylate protein samples obtained from embryonic and adult tissues as well as nervous system tumor cell lines. Phosphorylation status of GMFß was ascertained using primary glial tumors and Western immunoblotting assays. RESULTS: Both GMFß antibodies were specific but only anti-P02 recognized the candidate actin binding domain. Co-IP experiments confirmed GMFß and actin form protein complexes. Subcellular localization of GMFß changed with cytochalasin D but not colchicine administration, consistent with its role as an ADF. In primary embryonic mouse forebrain cultures and RA treated cells, GMFß was localized to axons and growth cones. Transfection of wild-type GMFß but not a C-terminal deletion mutant promoted process outgrowth. Phosphorylated GMFß (pGMFß) expression correlated with adult brain and low grade gliomas, such as pilocytic astrocytoma, whereas pGMFß was not predominantly expressed in embryonic brain or glioblastoma. CONCLUSIONS: Our results support a role for GMFß as a cytoplasmic protein that promotes process extension in vitro. GMFß binds directly to the actin cytoskeleton and is a member of the Actin Depolymerization Factor family. GMFß's phosphorylated form is highly expressed in the differentiated nervous system and low grade gliomas. Future studies will address whether GMFß and/or pGMFß expression correlates with survival in selected patient cohorts with low or high grade glioma. Furthermore, with the availability of a GMFß knockout mouse, the role of GMFß in glioma tumor invasion and signaling pathways can be addressed in vivo.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0127. RAPID AND FAITHFUL MODELING OF PEDIATRIC GLIOMA USING ELECTROPORATION-BASED SOMATIC TRANSGENESIS

Moise Danielpour 1, Rachelle Levy 1, C Danielle Antonuk 1, Javier Rodriguez Jr 1, Jessica Molina Aravena 1, Gi Bum Kim 1, David Gate 1, Serguei Bannykh 1, Clive Svendsen 1, Terrence Town 1, Joshua Breunig 1

Abstract

INTRODUCTION: Current pediatric brain tumor models rely on several types of methodologies, including engineered mouse lines, viral transduction, and cell transplantation. Each of these models has significant issues regarding either time for engineering and breeding in regards to mouse models, safety in the case of viruses, and natural progression of tumorigenesis in the case of transplantation. METHOD: We have created novel, autochthonic, in vivo models of low and high grade glioma. Specifically, we have combined postnatal electroporation with piggyBac transposition to mediate efficient and stable transgenesis of in vivo neural stem cells. Electroporation allows for an excellent degree of flexibility as multiple plasmids can be introduced simultaneously. In addition to the exquisite and flexible regulation of genetic determinants, electroporation gives us temporal and spatial control of the genesis of glioma. RESULTS: Preliminary results indicate that we can control the grade and ultimate tumor type by introduction of different combinations of genes. For example, RTK/Ras mutations typically lead to high grade oligodendroglioma. However, single Myc mutations have resulted in low grade tumor phenotypes. CONCLUSIONS: Electroporation allows for rapid gain- and loss-of-function genetic manipulation in the natural setting of neural stem/progenitor cells with restricted spatial transduction. It is hoped that this method will allow for substantial progress in the elucidation of the mechanisms of tumorigenesis in a wide range of glioma subtypes.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0128. UNCOVERING CLONAL EVOLUTION PATTERNS IN MEDULLOBLASTOMA METASTASES USING WHOLE GENOME SEQUENCING

A Sorana Morrissy 1, Chelsea Mayoh 2, Alan Lo 2, Nina Thiessen 2, Kane Tse 2, Richard Moore 2, Andrew Mungall 2, Xiaochong Wu 1, Timothy E Van Meter 3, Yoon-Jae Cho 4, V Peter Collins 5, Tobey J MacDonald 9, Xiao-Nan Li 10, Africa Fernandez-Lopez 6, David Malkin 8, Marco A Marra 2, Michael D Taylor 7

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant brain tumor in children. Although MB arises in the cerebellum, 40% of patients have leptomeningeal dissemination at diagnosis, a strong marker of poor prognosis. Despite surgery, whole brain and spinal cord radiation, and aggressive chemotherapy, patients with disseminated disease often have a poor outcome. Response to therapy can differ between the primary tumor and the metastases. The aims of our current study are (1) to understand the clonal evolution of disseminated disease from primary tumors, and (2) to identify metastasis-specific driver events that may provide targets for novel therapies. METHOD: We generated whole genome sequencing data from 12 children with matched germline, primary, and metastatic disease. Somatic aberrations were derived from this data, and 192 events (SNVs, indels) were selected from each patient for verification using deep amplicon sequencing. Selected variants included those detected in either the primary or metastatic tumors only, shared events displaying significant allelic frequency changes between primary tumor and metastasis, and all damaging mutations that could be identified. Deep sequencing at these loci (∼20,000X coverage) provides the necessary sampling depth to robustly identify changes in the clonal composition of the primary and metastatic tumors. RESULTS: The WGS data reveals shared mutations between the primary and metastatic tumors, confirming their clonal relationship. In the first patient studied, deep-sequencing identified metastasis-specific mutations confirmed to be absent in the primary tumor genome, indicating these arose de novo post-dispersal. Another subset of mutations, initially observed as metastasis-restricted n the WGS data, were identified at subclonal levels in the primary tumor. Together, these data support a model of dispersal in which a restricted clone in the primary tumor seeds metastases, and is undergoes further selection of existing and novel mutations. Mutations specific to the primary compartment are much less common. CONCLUSIONS: We have undertaken an investigation of allele-frequency data from matched primary and terminal metastatic tumors to establish the evolutionary history patterns of medulloblastoma. This disease is characterized by a low number of non-synonymous mutations, but notably, even at 30X WGS coverage, the majority of damaging mutations observed are enriched either in the primary or the metastatic compartments. Deep-sequencing data confirms that active clonal evolution is evident and affects the spectrum of damaging mutations in these disease compartments. This underscores the importance of studying metastatic disease, and the relevance of such an approach to the design of rationally applied targeted therapies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0129. PHASE II STUDY OF RAD001 IN CHILDREN AND ADULTS WITH NEUROFIBROMATOSIS TYPE 2 AND PROGRESSIVE VESTIBULAR SCHWANNOMAS

Matthias Karajannis 1, Genevieve Legault 1, Mari Hagiwara 1, Emilio Vega 1, Amanda Merkelson 1, Jeffrey Wisoff 1, John Golfinos 1, J Thomas Roland 1, Jeffrey Allen 1

Abstract

INTRODUCTION: Activation of the mammalian target of rapamycin (mTOR) signaling pathway is thought to be a key driver of tumor growth in Merlin (NF2) deficient tumors. Recent preclinical data indicate that differential activation of mTOR complex 1 (mTORC1) and mTORC2 may be cell-type dependent in NF2-deficient tumors and correlate with response to specific classes of mTOR inhibitors. RAD001 (everolimus) is an oral mTORC1-only inhibitor with anti-tumor activity in a variety of cancers. We conducted a single institution, prospective, open-label, two-stage phase II study to estimate the response rate to RAD001 in neurofibromatosis type 2 (NF2) patients with progressive vestibular schwannomas (VS). METHOD: Ten eligible patients were enrolled. RAD001 was administered at a daily dose of 10 mg (adults) or 5 mg/m2/day (children <18 years) PO in continuous 28-day cycles, for up to 12 cycles. Response was assessed every 3 months with MRI using 3-D volumetric tumor analysis, and audiograms. Nine patients were evaluable for the primary endpoint (one patient came off study after 3 weeks for personal reasons), defined as a ≥15% decrease in VS volume. Hearing response, defined as statistically significant increase in word recognition scores, was evaluable as a secondary endpoint in eight hearing patients. RESULTS: Three patients came off trial due to tumor progression after 3, 6 and 9 cycles, respectively. Three other patients discontinued treatment after 3, 6 and 6 cycles, respectively, because of lack of volumetric and/or hearing response. One patient completed 12 cycles, at which time he met criteria for hearing progression. One patient with stable disease is receiving his 11th cycle of treatment. One patient came off study after 3 cycles due to toxicity, i.e. pneumonia and decreased pulmonary function. All other observed toxicities were minor and expected. No objective imaging or hearing responses were observed and accrual was halted. CONCLUSIONS: None of the 9 patients with evaluable disease experienced an MRI or hearing response. Therefore, we conclude that RAD001 is ineffective for the treatment of progressive VS in NF2 patients. We are currently conducting a pharmacodynamic/pharmacodynamic ("Phase 0") study of RAD001 including pre-surgical VS patients to elucidate the biological basis for treatment resistance to mTORC1 inhibition in these tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0130. DOMINANT NEGATIVE OLIG2 IS INSUFFICIENT TO PREVENT GLIOMA INITIATION AND PROGRESSION

C Danielle Antonuk 1, Rachelle Levy 1, Gi Bum Kim 1, Terrence Town 1, Moise Danielpour 1, Joshua Breunig 1

Abstract

INTRODUCTION: Recent findings have suggested that Olig genes are essential for the proliferation of tumor cells and progression of glioma. Specifically, it has been documented that Olig genes are important for tumor cell proliferation and resistance to p53 responses to genotoxicity. METHOD: We have created a new model of high grade Oligodendroglioma, which displays ubiquitous expression of Olig2 and other oligodendrocyte markers. This model allows for co-expression of any plasmid. Employing this advantage, we employed a dominant negative form of Olig2 to assess the contribution of Olig2 repressor function to glioma initiation and propagation. RESULTS: Notably, expression of this DN-Olig2 failed to prevent tumor proliferation and invasion. Instead, the tumor cells converted from oligodendroglia-type progenitors into immature astroglial lineage tumor progenitors. CONCLUSIONS: These findings suggest that targeting Olig genes to treat tumors may be ineffective as gliomas can utilize Olig2 independent pathways for propagation by changing lineages. However, more investigation needs to be done regarding the possible function of Olig2 as a transcriptional activator in this context.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0131. IDENTIFICATION OF RESISTANCE MECHANISMS TO SMO INHIBITION IN MEDULLOBLASTOMA

Ekaterina Pak 1,2, Xuesong Zhao 1,2, Tatyana Ponomaryov 1,2, Rosalind Segal 1,2

Abstract

INTRODUCTION: The sonic hedgehog pathway (Shh) has been implicated in one-fourth of all medulloblastoma cases and thus provides a promising therapeutic target for these tumors. Most known inhibitors of the Shh pathway are selective antagonists of the pathway component, Smo. The first Smo antagonist was approved by the FDA last year. However, a major problem with these inhibitors is the emergence of drug resistance, for which the mutations and mechanisms are largely unknown. Our goal is to identify novel mechanism of resistance to Smo inhibition in medulloblastoma treatment and to develop ways of targeting tumors that have acquired resistance. METHOD: We have established a reliable in vitro model of Shh-dependent medulloblastoma that can be used to study resistance to Smo inhibitors. With this system, we are undertaking both targeted and unbiased approaches to identify mechanisms of resistance through either gain or loss of function. For our unbiased approach, we have implemented an in vitro forward genetic screen with transposon-mediated mutagenesis to identify resistant clones. To complement our in vitro studies and validate biological relevance, we are sequencing the exomes of resistant tumors that arise from in vivo xeonografts of Shh-dependent medulloblastoma cells. RESULTS: We verified that known mechanisms of resistance such as Gli2 over-expression, SuFu depletion, and expression of drug-resistant Smo mutations can confer resistance in our Shh-dependent medulloblastoma model. In the transposon-mediated screen, we have identified 18 resistant clones, among which 8 have insertions in SuFu, a known tumor suppressor downstream of Smo. By removing the transposon, we have demonstrated that mutations in SuFu are the cause of resistance to Smo inhibitors in these cells. We are now using this forward screen to identify other mechanisms of resistance. CONCLUSIONS: The results with SuFu validate our methodology for finding relevant resistance genes via transposon insertion. To identify novel resistance genes, we will expand the screen to attain additional non-SuFu resistant clones. Because Smo inhibitors are now in clinical trials for medulloblastoma, our efforts will elucidate potential mechanisms for therapeutic resistance, and will identify novel features of the Shh signaling pathway.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0132. A PIGGYBAC TOOLKIT FOR RAPID SOMATIC MUTATION AND CELL/TUMOR IMAGING IN THE CONTEXT OF IN VIVO PEDIATRIC GLIOMA

Rachelle Levy 1, C Danielle Antonuk 1, Jessica Molina Aravena 1, Gi Bum Kim 1, Clive Svendsen 1, Terrence Town 1, Moise Danielpour 1, Joshua Breunig 1

Abstract

INTRODUCTION: We have created a novel method for studying glioma initiation and progression in vivo-postnatal electroporation combined with piggyBac transposition. Unlike engineered mouse models or virally-derived glioma, electroporation allows for rapid addition of transgenes, simply by the inclusion of plasmid DNA in the initial injection. METHOD: Thus, we have created a toolkit for interrogating the resulting tumor cells in vivo. Specifically, we have created piggyBac plasmids expressing a spectrum of fluorescent proteins for labeling these cells in situ. Moreover, the latest in non-invasive genetic markers, including infrared proteins, luciferase, and a marker for use with magnetic resonance imaging has been cloned for this system. Finally, we are investigating the use of even more precise methods of genetic control, including inducible and reversible transgenic systems. RESULTS: These various imaging modalities have allowed for robust and flexible imaging of tumor cells. In particular, a membrane-tagged EGFP has allowed us to observe the rapid depletion of neural stem cells in our oligodendroglioma model. In addition, MRI imaging allows for precise non-invasive analysis of tumor progression. CONCLUSIONS: Here we discuss these methodologies and their use in the context of our novel pediatric glioma models.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0133. LONG-TERM OUTCOMES FROM A PROSPECTIVE PHASE II STUDY OF MULTI-AGENT SYSTEMIC AND INTRATHECAL CHEMOTHERAPY WITH AGE- AND RISK-ADAPTED RADIATION THERAPY FOR CHILDREN WITH NEWLY DIAGNOSED CNS ATYPICAL TERATOID/RHABDOID TUMOR (DFCI #02-294)

Susan Chi 1, Kenneth Cohen 2, Michael Fisher 3, Jaclyn Biegel 3, Daniel Bowers 4, Jason Fangusaro 5, Peter Manley 1, Anna Janss 6, Mary Ann Zimmerman 1, Mark Kieran 1

Abstract

INTRODUCTION: Atypical teratoid/rhabdoid tumor (ATRT) of the central nervous system (CNS) is a highly malignant neoplasm primarily affecting young children, with a historical median survival ranging between 6.5–10 months. Based on a small successful pilot series of patients with newly diagnosed and recurrent disease (Zimmerman, J Neuro-oncol, 2005), a prospective multi-institutional trial was conducted using a modified IRS-III regimen, including intrathecal chemotherapy, for patients with newly diagnosed CNS AT/RT (DFCI #02-294). The early results of this prospective trial were previously published (Chi, JCO, 2009). We now present the long- term survival outcomes for this regimen. METHOD: Treatment was divided into five phases: pre-irradiation induction chemotherapy (Weeks 1-6); chemo-radiation (Weeks 7-12); consolidation (Weeks 13 - 18); maintenance (Weeks 19 - 44); and continuation therapy (Weeks 45 - 51). Intrathecal chemotherapy administration alternated between the intra-lumbar and intra-ommaya routes. Patients with M0 stage received focal conformal radiation therapy (RT) at the prescribed time to a total dose of 5400 cGy. For patients over the age of 3 years with M+ disease at diagnosis, 3600 cGy craniospinal irradiation (CSI) was prescribed, with boost to primary sites of disease to total dose of 5400 cGy. RESULTS: Of 25 children enrolled, 22 were evaluable. Median age at diagnosis was 2.5 years (0.2 - 19.5 yrs). Twelve primary tumors were located in the supratentorial compartment, 10 in the posterior fossa. GTR of the primary tumor were achieved in 11 patients. Sixteen patients had M0 disease, one M2 and five M3. All patients received intrathecal chemotherapy. Seventeen patients received RT. Of the 14 patients evaluable for chemotherapeutic response, the objective RR was 62%, and from RT, 38%. The 3-year EFS and OS are 47% and 58 +/−12%, respectively, and the 5-year EFS and OS are 36% and 46 +/−13%, respectively. CONCLUSIONS: For this rapidly fatal disease, significant progress has been made in terms of improving and sustained survival. A future international study is planned to test the current backbone regimen against high-dose chemotherapy/autologous stem cell transplant, incorporating the growing biologic data on rhabdoid tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0134. RNA NANOPARTICLE VACCINES RE-DIRECT HOST-IMMUNITY AGAINST INTRACRANIAL MALIGNANCIES

Elias Sayour 1, Christina Pham 1, Luis Sanchez-Perez 1, David Snyder 1, Catherine Flores 1, Hanna Kemeny 1, Weihua Xie 1, Xiuyu Cui 1, Darell Bigner 1, John Sampson 1, Duane Mitchell 1

Abstract

INTRODUCTION: Malignant brain tumors are the number one cause of cancer death in children necessitating the development of targeted therapeutics. RNA-nanoparticle (RNA-NPs) can meet this need through delivery of total tumor RNA (ttRNA), targeting a broad repertoire of undefined and patient specific tumor antigens, to dendritic cells (DCs) inducing potent, nontoxic anti-tumor immunity. Since NPs have been used with limited toxicity in clinical-grade medicine, protect nucleic acids from degradation, and can be engineered to modulate immune responses, we have explored ttRNA-loaded NPs as an attractive "off-the-shelf" vaccine platform to target DCs in vivo thereby stimulating host-immunity to precipitate tumor efficacy. METHOD: The current study was undertaken to determine if vaccination with ttRNA-NPs would recruit DCs to effectively cross-prime immune responses generating anti-tumor efficacy in pediatric brain tumors without the induction of intolerable autoimmunity. To achieve this end, we investigated RNA-NP localization, DC trafficking, and their engagement of innate and specific immune responses using model antigens in TCR transgenic mice. Subsequently, anti-tumor efficacy was evaluated in intracranial tumor bearing mice vaccinated with NPs complexed with ttRNA extracted from murine medulloblastoma and glioma models. RESULTS: We screened commercially available and clinically translatable NP formulations and determined that the cationic liposome DOTAP was superior for delivery of RNA. Afterwards, we demonstrated the capacity to measure protein expression from RNA-NPs in vivo using a luciferase reporter assay and verified that RNA-NPs transfect DCs in vitro and in vivo. Furthermore, RNA-NPs were superior in the expansion of antigen specific T cells in vivo compared with positive control peptide vaccinations in complete Freund's adjuvant. Finally, we established that RNA-NPs generate anti-tumor efficacy in established intracranial tumors that can be potentiated with immunomodulatory RNAs encoding for GM-CSF. CONCLUSIONS: RNA-NPs are a novel platform for inducing potent nontoxic anti-tumor immunity in tumor bearing mice and may be harnessed to provide a more effective and specific therapy critical in improving clinical outcomes for children affected by malignant brain tumors without adding further toxicity to existing treatments. This data will establish the preclinical feasibility, efficacy, and toxicity data to support the rationale for clinical development of tumor RNA-loaded NP vaccines for recurrent medulloblastomas, and has a wide range of clinical applicability for all malignancies that can be targeting using ttRNA obtained from surgical resection of solid tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0135. BET BROMODOMAIN INHIBITION OF MYC-AMPLIFIED MEDULLOBLASTOMA

Pratiti Bandopadhayay 1, Brian Nguyen 2, Sabran Masoud 2, Nujsaubnusi Vue 2, Sharareh Gholamin 2, Furong Yu 2, Simone Schubert 2, Guillaume Bergthold 1, Siddhartha Mitra 2, Jun Qi 1, James Bradner 1, Mark Kieran 1, Rameen Beroukhim 1, Yoon-Jae Cho 2

Abstract

INTRODUCTION: Group 3 medulloblastomas are highly lethal tumors characterized by MYC-amplification. They are notably resistant to standard chemotherapy and radiotherapy, yet the development of targeted therapies against these tumors has been hampered by a lack of compounds that directly target MYC. It was recently discovered that drugs targeting bromodomains, protein regions that recognize acetylated lysine residues such as those found on N-terminal histone tails, effectively shut down MYC-associated transcriptional activity. JQ1 is a compound that inhibits BRD4, a BET bromodomain containing protein expressed in Group 3 medulloblastomas. Here, we investigate BET bromodomain targeting for the treatment of Group 3 medulloblastoma. METHOD: We tested the efficacy of JQ1 in established and newly generated patient- and GEMM-derived Group 3 medulloblastoma cell lines and xenografts, in vitro and in vivo. Non-MYC-amplified cell lines and normal human SVZ-derived neural stem cells were tested as controls. We performed cell cycle profiling and analyzed markers for apoptosis, senescence and differentiation after treatment with JQ1 versus control. The effect of JQ1 on MYC expression and global MYC-associated transcriptional activity was assessed by quantitative real time PCR and gene expression microarray profiling, respectively. In vivo efficacy of JQ1 was assessed in orthotopic xenografts established in immunocompromised mice. RESULTS: Treatment of MYC-amplified medulloblastoma cells with the active isomer of JQ1 (JQ1-S) resulted in decreased cell viability compared to treatment with the inactive enantiomer (JQ1-R). This corresponded with the down-regulation of MYC expression and MYC-associated transcriptional activity. JQ1-S treatment also resulted in a decrease in the percentage of cells transitioning through S phase compared to cells treated with JQ1-R. In contrast, no effect was observed with JQ1 on neural stem cells derived from the subventricular zone. We will present data from experiments which are currently underway examining the efficacy of JQ1 in in vivo models. CONCLUSIONS: JQ1 represses MYC expression and MYC-associated transcriptional activity in Group 3 medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-driven medulloblastomas. Furthermore, these data serve as rationale to rapidly move forward with early phase clinical trials examining the role of BET bromodomain inhibitors for children with these highly lethal tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0136. LONGITUDINAL CORTICAL THICKNESS CHANGES IN CHILDREN TREATED FOR MEDULLOBLASTOMA

Wilburn Reddick 1, John Glass 1, Qing Ji 1, Emily Paulus 1, Amar Gajjar 2, Robert Ogg 1

Abstract

INTRODUCTION: Children treated for medulloblastoma have neuroanatomical changes associated with neurocognitive deficits (IQ, attention, working memory, and processing speed). Based on a previous cross-sectional study reporting atypical cortical thinning in patients 1-8 years after therapy, it was anticipated that higher radiation doses to cortex around the posterior fossa and more distal cortical regions still undergoing maturation would exhbit the greatest thinning. This study explored longitudinal differences in cortical thickness in patients before, during, and following therapy. METHOD: Ten patients with medulloblastoma (7.6-20.3 years; 5 male; 6 average and 4 high risk) treated on the SJMB03 protocol (http://clinicaltrials.gov/ct2/show/study/NCT00085202) were imaged at baseline, 12 and 24 months, and 10 age-matched controls (7.6-20.7 years; 7 male) were imaged three times 12 months apart. High-resolution 3D T1-weighted MR imaging was acquired on a 3T whole-body system and processed with the FreeSurfer software (http://surfer.nmr.mgh.harvard.edu/) to assess cortical thickness. Statistical testing of cortical thickness differences between patients and controls was conducted at each time point using linear regression modeling with the QDEC package within FreeSurfer. All reported cortical differences were significant with P < 0.01. RESULTS: There was extensive cortical thinning in patients compared to controls at baseline, which may be attributed to disease, hydrocephalus, steroids, or surgery, but is present before either irradiation or chemotherapy. The extent of cortical regions with significant thinning decreased at 12 months and many regions were mostly resolved by 24 months. However, patients had some cortical areas which exhibited persistent thinning even 24 months after diagnosis. These regions included: bilateral superior frontal, right rostral middle frontal, left dorsal lateral frontal (pars orbitalis), bilateral superior and inferior parietal cortex, bilateral lateral occipital, left pericalcarine, and medial temporal. CONCLUSIONS: Children treated for medulloblastoma have significantly thinner cortex compared to their peers even before therapy begins. While cortical thinning resolves for most regions, there are select regions which are persistently thinner in patients. These cortical regions with persistent thinning have been associated with neurocognitive functions such as attention and memory. Assessing connectivity (eg, diffusion tensor imaging) within known or suspected functional networks that involve the regions with cortical abnormality may help define a neuroanatomical model to characterize the impact of therapy and treatment in this vulnerable population.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0137. QUIESCENT, THERAPY RESISTANT Sox2+ STEM CELLS PROPAGATE PATCHED1 +/- MOUSE MEDULLOBLASTOMA

Robert Vanner 1, Marc Remke 2, Tzvika Aviv 2, Lilian Lee 2, Xueming Zhu 2, Ian Clarke 2, Michael Taylor 2, Peter Dirks 2

Abstract

INTRODUCTION: Medulloblastoma growth and recurrence are driven by a subpopulation of cells with the stem cell property of unlimited self-renewal. In Sonic hedgehog (Shh) subgroup tumours, these cells' identity, and the mechanisms by which they propagate the tumour and resist therapy are poorly defined. We hypothesize that rare, therapy resistant Sox2+ stem-like cells propagate Shh-subgroup medulloblastoma. To test this hypothesis, we used the Patched1 +/- mouse model of medulloblastoma to evaluate stem (Sox2+), progenitor (DCX+) and neuronal (NeuN+) cell populations' proliferative dynamics in primary tumours. METHOD: Thymidine analogue pulse-chase and pulse-chase-pulse labeling of primary Patched1 +/- tumours was used to quantify cell populations’ proliferation and identify label-retaining cells. Apoptosis was assessed by IHC staining for active Caspase 3 and TUNEL. Intracranial infusion of the anti-mitotic Cytarabine (Ara-C) into Patched1 +/- mice ablated dividing cells. In vivo limiting-dilution analysis (LDA) of cells from primary Patched1 +/-; Sox2-eGFP tumours quantified the self-renewal potential of Sox2+ and Sox2- cells. Gene expression data from 64 primary medulloblastoma samples was obtained using the 4 × 44K feature Agilent Whole Human Genome Oligo Microarray. RESULTS: Chronic EdU pulse-chase experiments demonstrated the quiescent nature of Sox2+ cells versus the majority of tumour cells exhibiting rapid label acquisition and dilution. DCX+ progenitor-like cells are fast dividing, while NeuN+ cells are Ki-67-, yet labeled and lost EdU. Single EdU pulse-chase revealed NeuN+ cells are produced by differentiation of proliferating DCX+ cells. NeuN+ cells exhibit greater apoptosis than all tumour cells and Sox2+ cells. Sox2+ cells were confirmed tumour-initiating cells by LDA. Eliminating dividing cells enriched for Sox2+ cells, indicating therapy resistance. In human tumours, Sox2 is overexpressed within the Shh-subgroup where its expression negatively correlates with survival. CONCLUSIONS: For the first time, we have characterized self-renewal and proliferative heterogeneity in an in vivo brain tumour model. Our results suggest that Shh-subgroup medulloblastoma is propagated as an aberrant developmental process, where slowly dividing Sox2+ stem cells produce rapidly dividing progenitors that differentiate into short-lived neuronal cells. Sox2+ cells are therapy resistance due to their quiescent nature. The negative correlation between Sox2 expression and survival in human tumours points to the clinical relevance of these findings. Our results suggest targeting slowly dividing stem cells will be required for lasting medulloblastoma cures.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0138. IDH1 AND IDH2 MUTATIONS IN PEDIATRIC OLIGODENDROGLIOMAS

Ronald Hamilton 1, Ian Pollack 1

Abstract

INTRODUCTION: Introduction: Pediatric oligodendrogliomas are rare tumors. IDH1 and IDH2 mutations are common in adult oligodendrogliomas, but a recent study in 14 pediatric oligodendrogliomas found none with mutations. METHOD: Methods: A retrospective review (1960-present) of pediatric oligodendrogliomas and low-grade tumors at our institution. After neuropathological review and application of current diagnostic criteria, 36 pediatric oligodendrogliomas were identified as well as 20 other low-grade gliomas with oligodendroglial-like features (pilocytic and cerebellar neoplasms were excluded). These formalin-fixed paraffin embedded cases were microdissected and evaluated for IDH1 (R132H and R132G) and IDH2 mutations by PCR. RESULTS: Results: In the 36 pediatric oligodendrogliomas, 3 cases showed mutations of IDH1 and none with mutations in IDH2. The IDH1 mutations included 2 R132H and 1 case with the rare variant R132G. None of the other low grade tumors had any mutations in IDH1 or IDH2. CONCLUSIONS: Conclusions: In the largest pediatric oligodendroglioma cohort examined to date we found that markedly fewer pediatric oligodendrogliomas have IDH mutations compared to adult cohorts. In one case the recurrent tumor, but not the primary, showed IDH1 mutation, suggesting that IDH1 is part of the oncogenesis of these tumors in children. IDH1 mutations were not found in other pediatric low grade gliomas in our cohort.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0139. DEVELOPMENT OF COMBINED PK/PD STUDIES OF RAF/MEK/mTOR INHIBITORS FOR THE TREATMENT OF PEDIATRIC LOW-GRADE ASTROCYTOMAS BY MALDI MASS SPECTROMETRY IMAGING

David Calligaris 1, Xiaohui Liu 1, Daniel Feldman 1, Christopher Thompson 4, Jennifer Ide 1, Sara Buhrlage 3, Nathanael Gray 3, Mark Kieran 2, Charles Stiles 3, Nathalie Agar 1

Abstract

INTRODUCTION: Pediatric low-grade astrocytomas (LGAs) account for the majority of pediatric central nervous system neoplasms. A wealth of studies has evidenced disregulations of the MAPK pathway in tumorigenesis of such tumors. This pathway is a conserved signaling cascade involving various protein kinases for signal transduction from the cell membrane to the nucleus to mediate biological functions such as cell growth, survival, and differentiation. MALDI mass spectrometry imaging has proven to be an effective method to reveal the two-dimensional spatial distribution of drugs, lipids and peptides in tissue, and opens the way to potentially combine pharmacokinetic and pharmacodynamic studies of RAF/MEK/mTOR inhibitors. METHOD: MS and MS/MS analyses of serial dilutions of RAF/MEK/mTOR inhibitors were performed from solutions spotted directly on target and on mounted mouse brain tissue sections with different matrices using an UltrafleXtreme MALDI TOF/TOF instrument. 10 µm thickness brain and liver sections from pathway relevant orthotopic mouse models treated with each of eight compounds were used for MALDI MSI. Drug distribution was imaged from treated liver/brain sections using MALDI TOF/TOF or SolariX FTICR (12T). For proteomics, brain sections were enzymatically digested with trypsin, and imaged by MALDI TOF. For lipidomics, brain sections coated with 1,5-DAN were analyzed by MALDI FTICR MSI. RESULTS: The MS analysis on the drug standards allowed us to optimize parameters for their analysis by MALDI MSI. Using heme as a natural marker of the vasculature, MALDI MSI analyses allowed visualization of relative blood-brain barrier (BBB) penetrance of some of the investigated RAF/MEK/mTOR inhibitors and/or their metabolites. Proteomic and lipidomic profilings of brain sections are in progress for a subset of the orthotopic mouse models treated with BBB penetrating drugs to assess tumor response to targeted treatment. CONCLUSIONS: The presented pharmacoproteomics and pharmacolipidomics data combined with the study of drug distribution have the potential to provide a valuable advance for the identification of drug-responsive biomarkers in the development and implementation of novel and more effective therapeutic approaches. This study evidences the main input of MALDI MSI in drug re-purposing and development for the treatment of brain cancers.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0140. DISSECTING MOLECULAR AND PROGNOSTIC SUBTYPES OF GROUP 3 AND GROUP 4 MEDULLOBLASTOMAS

Marc Remke 1, Florence MG Cavalli 1, Paul A Northcott 2, Marcel Kool 2, Stefan M Pfister 2, Michael D Taylor 1, MAGIC Project 3

Abstract

INTRODUCTION: Integrated genomics approaches have revealed distinct genetics, transcriptomics, and prognosis of the four medulloblastoma subgroups. Clinically, Group 3 and Group 4 are characterized by frequent metastatic dissemination and an unfavorable prognosis. These tumors exhibit a remarkable clinical and biological intertumoral heterogeneity. Early evidence suggests that subtypes exist within Group 3 and Group 4, potentially accounting for this heterogeneity. Previous attempts to identify the underlying biology driving these subtypes, and to define prognostic subtypes have been underpowered because of small samples sizes included in earlier genomic studies. METHOD: To elucidate these subtypes, we performed a high-resolution genome-wide methylation profiling analysis using the Infinium HumanMethylation450 BeadChip in a discovery set of 361 frozen primary Group 3 or Group 4 medulloblastomas. We carried out comprehensive genome-wide copy number and gene expression analysis for matching samples. Unsupervised consensus clustering approaches were used to identify biological subtypes for each of the datasets. We integrated information across platforms using integrative clustering approaches, and delineated key driver genes and pathways using bioinformatic algorithms. Subtypes were correlated with clinicopathological information. RESULTS: Unsupervised consensus clustering analyses of transcriptomic and methylation data revealed three distinct subtypes within both Group 3 and Group 4. Subtype-specific distributions of clinicopathological, genomic, epigenomic and transcriptional characteristics were elucidated using an integrated approach. Poor prognosis was observed in a Group 3 cluster enriched in LCA histology, TGF-beta pathway deregulation, and MYC amplification. MYC status or M stage had no prognostic impact within this cluster. Group3 infants defined an intermediate risk group. Intriguingly, a cluster comprising one third of Group 3 tumors delineated patients with a favorable prognosis, who would be considered high-risk based on current molecular stratification schemes. CONCLUSIONS: Integrated analyses of transcriptomic and genomic alterations delineate distinct subtypes within Group 3 and Group 4. SNCAIP duplications and diploidy defined one Group 4 subtype, while the other clusters showed tri-/tetraploidy, and MYCN or CDK6 amplifications, respectively. Future research on medulloblastoma and the development of clinical trials should take into consideration these distinct subtypes of medulloblastoma to improve risk stratification, and to define future avenues for rational, targeted therapy.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0141. FUNCTIONAL ANALYSIS OF THE H3.3-K27M MUTATION IN DIPG

Patricia Rakopoulos 1, Sanja Pajovic 3, Pawel Buczkowicz 1, Andrew Morrison 3, Eric Bouffet 3, Ute Bartels 3, Oren Becher 2, Cynthia Hawkins 3

Abstract

INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is a rare but devastating pediatric brainstem tumor. It is usually diagnosed based on clinical symptoms and radiological detection of a tumor mass centrally located in the pons. Up to now efforts to treat this tumor have been unsuccessful. Our lab has been studying the genetics of DIPG with the hope of developing more effective treatment. Recent whole genome sequencing efforts by our lab and others have identified a highly recurrent K27M mutation in the histone variant gene H3F3A. Here we investigate the potential function of H3.3-K27M in tumorigenesis. METHOD: To investigate the role of H3.3-K27M in the initiation and/or maintenance of DIPG, we introduced the mutation into various cell lines including normal human astrocytes (NHAs) and neural stem cells (NSCs). To assess genome-wide changes in gene transcription we performed HT12 gene expression analysis using H3.3-K27M and WT- H3.3 over-expressing NHAs. Additionally, we tested the ability of these cells to grow in an anchorage independent manner and form tumors in the brainstem of immunocompromised mice. Immunohistochemistry of DIPG patient samples was performed to assess the global effect of H3.3-K27M on histone marks associated with activation and/or repression of gene expression. RESULTS: We observed that over-expression of H3.3-K27M altered cell growth of NHAs as well as the sphere-forming potential of NSCs. Gene expression profiling of NHAs over-expressing H3.3-K27M or WT-H3.3 revealed widespread changes in gene expression affecting multiple pathways. Interestingly, these cells did not form colonies in an anchorage independent assay and have yet to form tumors in mice. Immunohistochemistry of human DIPG tissue suggests that H3.3-K27M positive tumors have altered levels of H3K27 histone marks. CONCLUSIONS: Our data suggests that while H3.3-K27M over-expression is able to affect cell growth and gene expression, it is unable to completely transform NHAs on its own. The effect of H3.3-K27M appears to be highly dependent on the cellular context and as such, future work will aim to elucidate how cooperation with other gene mutations, like those in p53, affect the tumorigenic potential of these cells.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0142. THE TYPE OF HISTONE H3 VARIANT K27M MUTATION DRIVES THE AGRESSIVENESS OF DIFFUSE INTRINSIC PONTINE GLIOMA

Nathalene Truffaux 1, Stephanie Puget 2, Cathy Philippe 1, David Castel 1, Katy Taylor 3, Alan Mackay 3, Ludivine Le Dret 1, Patrick Saulnier 1, Raphaël Calmon 2, Nathalie Boddaert 2, Thomas Blauwblomme 2, Christian Sainte-Rose 2, Chris Jones 3, Jacques Grill 1

Abstract

INTRODUCTION: DIPG is the most challenging brain tumor for biologists and oncologists. No significant improvement in the prognostication, diagnosis or treatment of this tumor has been made in the last 30 years. Recent high-throughput sequencing data have shown the frequent occurence of mutations in the histone variants H3.3 (H3F3A) or H3.1 (HIST1H3B) but little is known of the biological changes accompanying these mutations. METHOD: The type of histone H3 variant mutations were characterized by Sanger Sequencing in 140 pediatric high-grade gliomas including 51 DIPG confirmed radiologically and systematically biopsied at diagnosis. Mutations were correlated with histological (oligodendroglial vs astrocytic), immunohistochemical (EGFR, P53, Olig2, MIB1), genomic (specific amplifications or losses), transcriptomic (mesenchymal or proneural) and survival data. Supervised clustering of gene expression according to the type of mutation was also performed. RESULTS: Mutations at K27M in histone H3 variants were found in 45/51 (88%) of DIPG. The K27M mutation was seen twice more frequently in H3F3A. While mutations in H3F3A were also present in thalamic or medullary gliomas, the mutations of HIST1H3B were only seen in DIPG. The type of histone H3 variant mutated was not linked with histology or GE profiling in DIPG. Amplification of PDGFRA was more frequent in H3F3A (23% vs 4%, p = 0.05463). Overall survival was significantly shorter in patients with H3F3A-mutated DIPG (median OS = 11 months vs 15 months, p = 0.03). CONCLUSIONS: Almost all true DIPG harbor a K27M mutation in one of the two histone H3 variants. Our data do not show that the type of histone H3 variant K27M mutation is driving a specific gene expression profile or phenotype; however it may influence the behaviour of the tumor. Indeed, mutations in H3F3A characterize rapidly progressing DIPG. If confirmed in follow-up studies, this could be one of the first prognostic marker to be taken into account when judging the results of prospective trials.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0143. VALIDATION OF HEME AS A MARKER OF THE VASCULATURE FOR MALDI IMAGING OF THE BLOOD-BRAIN BARRIERS IN DRUG DEVELOPMENT

Xiaohui Liu 1, Maritza Ebling 2, Jennifer Ide 1, Lan Wang 2, Erin Davis 2, Mark Marchionni 2, Darrin Stuart 4, John Alberta 2, Mark Kieran 3, Charles Stiles 2, Nathalie Agar 1

Abstract

INTRODUCTION: Development of drugs for brain tumor treatment is challenged by methodologies used to assess the ability of drugs to penetrate the blood-brain barrier (BBB). The blood-brain barrier is a natural protective boundary for the human brain from the entrance of exogenous toxins as well as potential drugs. Therefore, many drugs that target other cancers are not effective for brain tumors. Determining drug permeability is critical for brain cancer drug development. Using mass spectrometry imaging (MSI), we used heme as a natural marker of brain vasculature and applied it to interpret the drug permeability to brain tumors. METHOD: Fluorescein and fluorescein isothiocyanate (FITC) were injected in mice through tail vein injection. For drug analysis, the mice were dosed with RAF265 and BKM120 through oral gavage. The mouse organs were flash frozen after dissection and the brain tissues were sectioned to 10 µm thickness. 2,5-Dihydroxybenzoic acid was prepared and deposited using the ImagePrep (BrukerDaltonics, Germany). Mouse brain sections were imaged by fluorescence microscope followed by MALDI TOF/TOF. Tissue sections were imaged with the UltrafleXtreme MALDI TOF/TOF (BrukerDaltonics, Germany). Tissue sections were imaged at 20-150 µm spatial resolution. RESULTS: The fluorescence images of fluorescein and FITC were correlated with the heme and fluorophore images obtained from MALDI MSI, validating heme as an effective marker to visualize the blood-brain barrier. RAF265, known to be effective in sub-cutaneous animal models, but not in orthotopic brain models, was observed to accumulate in the tumor region and ventricles. However, most RAF265 in the tumor region was co-localized with heme signals. BKM120, which has high permeability, was observed to distribute more evenly throughout the brain without co-localizing with heme. CONCLUSIONS: Our experiments validated MALDI MSI to be an effective approach to image the blood-brain barrier using heme as the surrogate and investigate drug permeability to the tumor in a way that can significantly inform and guide drug re-purposing and development. The co-registration of RAF265 with heme signals in tumor region demonstrates that the majority of the drug dose is retained within the inefficient blood capillaries of the tumor, and not accessing the tumor tissue. However, BKM120 does not accumulate in the tumor region with high drug penetrance and spreads out across the entire brain.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0144. INTEGRATIVE CLUSTERING REVEALS FOUR CLINICALLY AND MOLECULARLY DISTINCT SUBTYPES OF SHH MEDULLOBLASTOMA

Marc Remke 1, Florence MG Cavalli 1, Paul A Northcott 2, Marcel Kool 2, Stefan M Pfister 2, Michael D Taylor 1, MAGIC Project 3

Abstract

INTRODUCTION: Sonic hedgehog (SHH) medulloblastomas account for one-third of all patients and comprise the majority of infant and adult medulloblastomas. Although all tumors in the SHH subgroup show over-activity of SHH pathway, this subgroup exhibits remarkable heterogeneity in its transcriptional signatures, somatic copy number alterations, and mutations. To date, few genomic studies have profiled sufficient cases to identify distinct molecular subtypes within this subgroup, making it difficult to define molecular targets for the development of novel treatment strategies. METHOD: To specifically address these issues, we performed comprehensive genome-wide gene expression analysis using the Affymetrix GeneChip Human Gene 1.0 ST Array and a high-resolution methylation profiling analysis using the Illumina Infinium HumanMethylation450 BeadChip in a discovery set of >250 primary SHH medulloblastomas. Unsupervised clustering was performed to delineate molecular subtypes for each of the datasets. Integrative clustering was used to incorporate profiling data from different platforms. Subtype-specific pathway analysis was carried using Ingenuity and Cytoscape. RESULTS: Unsupervised consensus clustering analyses of transcriptomic and methylation data revealed four distinct subtypes. SHH tumors reveal distinct methylation and expression signatures in infant and adult tumors further underlining age-specific difference in SHH tumor biology. Notably, both infant and adult SHH tumors can be further separated into two distinct clusters including different proportions of pediatric and adolescent patients. Subtype-specific distributions of clinicopathological and molecular characteristics were elucidated using an integrated evaluation of the genome, epigenome, and transcriptome. Integrative clustering revealed a subset of SHH tumors characterized by 10q loss, specific gene expression and CpG methylation changes with a particularly poor prognosis. CONCLUSIONS: Collectively, our integrated analysis demonstrates that pediatric and adult SHH medulloblastomas are clinically, epigenetically, transcriptionally, genetically, and prognostically distinct. A refined understanding of the subtypes within infant and adult SHH tumors may help to develop novel outcome prediction algorithms and define novel targets for molecular-based therapies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0145. GLIAL TUMOR ORIGINS IN A BRAFV600E -DRIVEN MOUSE MODEL OF PEDIATRIC GLIOMA

An-Chi Tien 1, Amelie Griveau 1, David Rowitch 1

Abstract

INTRODUCTION: Malignant astrocytoma is a leading cause of brain tumor related mortality in children. Recently we have shown that the activating mutations of BRAF, a member of the serin/threonine protein kinases, are present in ∼20% of pediatric glioma gr. II-IV. In addition, concomitant homozygous deletion of CDKN2A, encoding tumor suppressor protines p14 and p16, was observed in 71% of such BRAFV600E positive gliomas. METHOD: To generate a de novo mouse model bearing similar genetic mutations, we used hGFAP-cre that is expressed in astrocyte as well as oligodendrocyte precursor cells (OPC), to activate BRAFV600E in a cdkn2a null genetic background. RESULTS: We have previously shown that these mice over-produce astrocytes in spinal cord during fetal-early post-natal stages (Tien et al., 2012). In contrast, we report ongoing proliferation of OPC is persistent until around P21 when mice become neurologically symptomatic. We then treated these mice with a mutant BRAFV600E specific inhibitor PLX4720. By using 20mg/kg/day regime, the OPC population and the proliferation index revealed by ki-67 staining was significantly reduced. Currently, we are directly testing OPCs as a possible cell-of-origin through FACS purification of NG2+ cells. CONCLUSIONS: These findings will be used to determine competence of OPCs to give rise to BRAF-driven glioma and effects of BRAF small molecule inhibitors on this process.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0146. GENOMIC ANALYSIS OF DIFFUSE PEDIATRIC LOW-GRADE GLIOMAS IDENTIFIES RECURRENT, ONCOGENIC MYBL1-TRUNCATING REARRANGEMENTS

Lori Ramkissoon 1, Peleg Horowitz 2, Justin Craig 1, Shakti Ramkissoon 2, Benjamin Rich 1, Guillaume Bergthold 1, Uri Tabori 8, Hala Taha 7, Daniel Bowers 9, Cynthia Hawkins 8, Roger Packer 10, Charles Eberhart 5, Liliana Goumnerova 4, Jennifer Chan 11, Sandro Santagata 2, Scott Pomeroy 4, Azra Ligon 2, Mark Kieran 4, Rameen Beroukhim 3, Keith Ligon 1

Abstract

INTRODUCTION: Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children. While unifying genetic events have been identified in some PLGG subtypes, including BRAF duplications in pilocytic astrocytomas BRAF V600E mutations in pleomorphic xanthoastrocytomas and gangliogliomas, the rarity and diversity of diffuse PLGGs has historically impeded determination of genetic driver events specific to these tumors. Identification of novel genetic alterations in diffuse PLGGs would increase biologic understanding of tumor behavior as well as define diagnostic molecular subclasses. METHOD: Using a novel method for reliable performance of array comparative genomic hybridization (aCGH) on formalin-fixed paraffin embedded (FFPE) archival samples, we performed high-resolution copy-number analysis on 44 diffuse PLGGs to determine recurrent alterations. We then subjected two representative diffuse astrocytomas grade II with different copy number profiles for further characterization by whole genome sequencing. Functional studies were also carried out to determine the oncogenic potential of novel, recurrent events. RESULTS: We identified significantly recurrent events including 8q13.1 gains in 28% of DA2s. These alterations resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar deletion-truncation breakpoint was identified in an angiocentric glioma in the related gene MYB. Whole-genome sequencing (WGS) of a DA2 demonstrated MYBL1 tandem duplication. Two novel, truncated MYBL1 transcripts isolated from this tumor induced anchorage-independent growth when expressed in 3T3 cells and formed tumors in mice. WGS of a MYBL1-wild-type DA2 uncovered TP53, ATRX, and H3F3A mutations previously reported only in pediatric glioblastoma; this DA2 subsequently progressed to glioblastoma. CONCLUSIONS: Our data identify several recurrent somatic genetic events in PLGG, some of which track with specific histologic types. The most significant of these was recurrent focal amplification of MYBL1, found exclusively in DA2 samples, none of which had other PLGG-associated lesions such as BRAF duplication. Focal deletion of 6q involving the MYB locus was also observed in one angiocentric glioma in our dataset, similar to deletions in two individual cases of angiocentric glioma noted in prior studies. Taken together our results highlight a potential role for the MYB family in the biology of low-grade gliomas.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0147. GENERATION OF ANTI-PDGFRα IMMUNOTOXINS FOR BRAINSTEM GLIOMA TREATMENT

Chien-Tsun Kuan 1, Vidyalakshmi Chandramohan 1, Stephen Keir 1, Ira Pastan 2, Darell Bigner 1

Abstract

INTRODUCTION: Despite radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for effective therapeutic strategies to improve survival. Platelet-derived growth factor receptor alpha (PDGFRa) is identified as a potential therapeutic target in whole-genome profiling of pediatric brainstem glioma. Recombinant immunotoxins (RITs) are composed of an Fv that binds to an antigen on a cancer cell surface fused to a toxin, thus killing only the cancer cells. We have generated an RIT that targets PDGFRa for treating these highly malignant tumors. METHOD: We have constructed an RIT, PD1-PE38KDEL, by fusing an anti-PDGFRa scFv, 2.449.1.3 VH-(G4S)3-VL, with a truncated form of Pseudomonas exotoxin A, PE38KDEL, which carries a C-terminal KDEL peptide for improved intracellular transport. PD1-PE38KDEL was expressed in E. coli, and the inclusion bodies were reduced, refolded, and purified by ion exchange chromatography and gel filtration. The cytotoxicity of PD1-PE38KDEL was measured by protein inhibition assay on PDGFRa-transfected cells and pediatric GBM cells, D930MG and D717MG. In vivo anti-tumor activity was conducted by intraperitoneal injection of PD1-PE38KDEL at 3 doses of 0.3 mg/kg or 0.6 mg/kg in a subcutaneous human xenograft model. RESULTS: The PD1-PE38KDEL protein was purified as a monomer of 64 kDa to a homogeneity of 95% by SDS-PAGE. We tested the in vitro activity of PD1-PE38KDEL on various pediatric glioma xenograft cells and demonstrated cell-killing activity. The IC50 of PD1-PE38KDEL on PDGFRa-transfected cells was 5 ng/mL, and on D930MG pediatric GBM cells, it was 10 ng/mL; The IC50 was 40 ng/mL on D717MG cells. There was no cytotoxicity of anti-Tac-PE38KDEL, a negative control, on PDGFRa-positive cells. Relative to the control group, the PD1-PE38KDEL-treated mice showed a statistically significant (P = 0.015) growth delay, with a T − C of 5.4 days. CONCLUSIONS: The RIT PD1-PE38KDEL showed potent cytotoxicity against PDGFRa-transfected cells and the cells from human pediatric GBM xenografts. The median survival of treated animals was 5.4 days longer than the control group (P = 0.015). Our results demonstrate that PD1-PE38KDEL is efficacious in targeting the brainstem tumor surface antigen PDGFRa. Further investigations of this RIT as a treatment for brainstem glioma will be pursued in a convection-enhanced delivery animal model. This RIT not only has significant therapeutic potential for brainstem glioma, but for other PDGFRa-expressing malignancies as well.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0148. USING MAGNETIC RESONANCE IMAGING TO DETERMINE GADOLINIUM CONTRAST AGENT CONCENTRATION

Zhiping Zhou 1, Sharon Ho 1, Henning Voss 1, Mark Souweidane 1

Abstract

INTRODUCTION: Convection-enhanced delivery (CED) has the ability to deliver drugs, including macromolecules, into the brain with high concentration by bypassing the blood-brain barrier, which makes it a promising drug delivery method in the treatment of brain tumors including diffuse intrinsic pontine glioma. However, there are currently no adequate methods to evaluate in vivo drug distribution and local concentration in CED. This is analogous to not knowing pharmacokinetics in systemic delivery or dose and fields in radiation therapy. We studied the feasibility of using Gd-DTPA as a surrogate tracer in CED and using magnetic resonance imaging (MRI) to determine its concentration distribution. METHOD: Gd-DTPA standards (12 concentrations) were used to optimize scanning sequences for longitudinal relaxation time (T1) and relaxivity (R1) on a Bruker 7.0 Tesla scanner. T1 were determined by using the following relationship: S = gf(T1), where S is the signal intensity, g is the scaling coefficient and f(T1) is a function dependent on the imaging sequence. Standard curve was obtained by fitting R1 values to a linear model. Then hydrogel phantoms were infused with Gd-DTPA and scanned with the optimized sequence to determine Gd-DTPA concentration. RESULTS: In Gd-DTPA standards, T1 values were mapped for each concentration and R1 values were deduced. Gd-DTPA concentrations and R1 values follow an excellent linear relationship. Using this linear relationship, Gd-DTPA concentrations in the hydrogels at different spatial locations and time points after infusion were determined. CONCLUSIONS: Gd-DTPA concentration can be determined using R1 values from MRI scans. Future studies will apply this method to in vivo determination of Gd-DTPA concentration, which will require compensating for the difference in longitudinal relaxivities of Gd-DTPA in hydrogel and brain tissues. The results may improve CED by allowing clinicians to monitor and sustain agent concentrations at target sites for the treatment of brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0149. PATTERNS OF PROGRESSION IN PEDIATRIC PATIENTS WITH HIGH-GRADE GLIOMA OR DIFFUSE INTRINSIC PONTINE GLIOMA TREATED WITH BEVACIZUMAB-BASED THERAPY AT DIAGNOSIS

Ralph Salloum 1, Mariko DeWire 1, Maryam Fouladi 1, Stewart Goldman 2, Lionel Chow 1, Trent Hummel 1, Kathleen Dorris 1, Lili Miles 1, Mary Sutton 1, Charles Stevenson 1, James Leach 1

Abstract

INTRODUCTION: Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor, is FDA-approved for adults with recurrent glioblastoma multiforme (GBM) and is undergoing clinical trials in adults and children with newly-diagnosed high-grade gliomas (HGG), including diffuse intrinsic pontine glioma (DIPG). Sixty percent to 75% of adult GBM patients receiving bevacizumab at diagnosis or recurrence have been reported to have a "diffuse invasive" pattern of progression believed to be due to the development of "evasive resistance" to anti-angiogenenic therapy. There is a paucity of data regarding patterns of progression in children with HGG/DIPG treated with bevacizumab-based therapy at diagnosis. METHOD: We performed a retrospective study of children with HGG or DIPG who received bevacizumab-based therapy at initial diagnosis. Patients received radiotherapy with bevacizumab followed by maintenance bevacizumab, irinotecan ± temozolomide on or according to study at Cincinnati Children's Hospital Medical Center (CCHMC). Seventeen patients < 22 years were included. We reviewed magnetic resonance imaging (MRI) characteristics of first and most recent progression . Imaging definitions of progressive disease (PD) were local: at primary site or within 2 cm, contiguous; diffuse: > 2 cm away but contiguous with primary site, ill -defined and infiltrative; distant: new, non-contiguous enhancing or non-enhancing disease. RESULTS: Fourteen children had DIPG, 3 had HGG. Median age at diagnosis was 7 years (range: 3-21). Median time to PD and median follow-up were 8.2 months (range 3.5-21) and 9 months (range: 4-24), respectively. Among 12 patients with PD, 9 (75%) had local and 3 (25%) had local, diffuse and distant PD, at initial progression. Patterns of distant PD included hemispheric periventricular (n = 2; DIPG) and non-contiguous hemispheric PD (n = 1; HGG). At the most recent PD at a median time of 10 months (range 7-14) from diagnosis, 6 of 12 (50%) had at least diffuse (n = 4), or distant (n = 5) PD. CONCLUSIONS: In newly-diagnosed children with HGG or DIPG who received bevacizumab-based therapy, a diffuse or distant pattern of progression was noted in 25% at first progression, and 50% at the most recent progression. This is the first pediatric study describing patterns of progression in newly diagnosed HGG and DIPG patients receiving bevacizumab-based therapy. The high incidence of diffuse or distant progression may be explained by resistance to anti-angiogenic therapy through activation of alternative pro-angiogenic pathways or co-option of native blood vessels which can lead to increased invasiveness.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0150. DISTINCT IMMUNE CELL INFILTRATE PROFILES ACROSS PEDIATRIC BRAIN TUMOR TYPES

Andrea Griesinger 1, Andrew Donson 1, Lindsey Hoffman 2, Diane Birks 1, Vladimir Amani 1, Michael Handler 2, Nicholas Foreman 2

Abstract

INTRODUCTION: Emerging evidence exists for anti-tumor immune control in the central nervous system. Microarray-based studies by our laboratory confirm that the immune control of tumor progression extends to the “immunoprivileged” central nervous system, identifying prognostic immune gene signatures in primary tumor specimens. Using a more direct multicolor flow cytometric approach we have systematically characterized the phenotype, frequency and function of infiltrating immune cells across the spectrum of pediatric brain tumor types and normal brain. METHOD: A panel of dissagregated primary pediatric brain tumor samples (15 ependymoma (EPN), 4 high grade glioma (HGG), 6 pilocytic astrocytoma (PA) and 6 medulloblastoma (MED)) and 5 normal brain samples obtained from epilepsy patients were analyzed by flow cytometry. Immune cell types analyzed included both myeloid and lymphoid lineages and their respective markers of polarization toward M1/M2 and/or TH1/TH2 functional phenotypes. RESULTS: Immune parameters that distinguished each of the tumor types were identified. EPN and PA demonstrated significantly higher infiltrating myeloid and lymphoid cells compared to HGG, MED or normal brain. Both EPN and PA demonstrated an M1 functional phenotype compared to normal brain and MED. However, EPN was unlike PA in that it showed a TH2 functional phenotype. Similar to normal brain, HGG and MED each exhibited M2 functional phenotypes markers and neither contained a clear TH1/2 phenotype status, possibly due to limited cell numbers. In all tumors, CD4 and CD8 T-cells overexpressed CD45R0 compared to normal brain. CONCLUSIONS: These results of this comparative tumor analysis demonstrate that different pediatric brain tumor types exhibit distinct immune microenvironments. This suggests that individual immunotherapeutic approaches need to be designed for each tumor type. Furthermore, the immune characteristics identified in this study can be manipulated in order to optimize the efficacy of such immunotherapies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0151. PD0332991, a cdk4/6 INHIBITOR, INDUCES TUMOR REGRESSION AND EXTENDS SURVIVAL IN ATYPICAL TERATOID RHABDOID TUMOR (ATRT) AND MEDULLOBLASTOMA MOUSE MODELS

Michelle Cook Sangar 1, Kyle Pedro 1, Sally H Ditzler 1, Emily Girard 1, James Olson 1

Abstract

INTRODUCTION: The cyclin dependent kinases 4 and 6 (cdk4/6)-cyclin D-INK4-retinoblastoma protein signaling pathway is frequently deregulated in cancer and has potential to be an important target in treatment of atypical teratoid rhabdoid tumors (ATRTs) and medulloblastomas. The vast majority of ATRTs have biallelic inactivating mutations in SMARCB1, which causes cell cycle progression by downregulation of p16INK4A and upregulation of cyclin D and E2Fs. Likewise, cyclin D expression is increased in most medulloblastomas and is a poor prognostic indicator. Since these pathway aberrations lead to increased activity of cdk4/6, we assessed efficacy of a cdk4/6 inhibitor in ATRT and medulloblastoma mouse models. METHOD: The efficacy of PD0332991, a specific inhibitor of cdk4/6, was investigated using patient-derived xenotransplant (PDX) mouse models of ATRT and medulloblastoma, generated using freshly resected tumor tissue from pediatric patients at Seattle Children's Hospital or Children's Oncology Group (COG) sites. PD0332991 (150 mg/kg or 75 mg/kg) was administered daily by oral gavage. Efficacy was assessed by measuring the difference in tumor size (subcutaneous xenografts) or survival (orthotopic xenografts) in drug-treated as compared to vehicle-treated mice. Pharmacodynamic analyses involved investigation of the phosphorylation state of Rb protein and the extent of Ki67 positive staining using western blot analysis and immunohistochemistry. RESULTS: PD0332991 treatment caused significant regression of subcutaneous ATRT and medulloblastoma tumors as compared to vehicle treatment. Rb phosphorylation at Ser780 was significantly reduced in drug-treated tumors. Likewise, Ki67, a marker of cell proliferation, was significantly reduced following drug treatment. In each of 3 survival studies using orthotopic medulloblastoma models, all vehicle-treated mice (n = 12-13) died, while all drug-treated mice (n = 11-13) survived for the study duration (10-28 days). In the ATRT orthotopic mouse model, 13/17 drug-treated mice were alive at the end of the study (45 days) as compared to 1/17 vehicle-treated mice. There was no apparent toxicity associated with drug treatment. CONCLUSIONS: Inhibition of cdk4/6 is a promising therapeutic strategy for both ATRTs and medulloblastomas. Treatment with PD0332991 resulted in significant tumor regression in subcutaneous tumor models and a highly significant survival advantage in orthotopic models. Pharmacodynamic analyses indicate that PD0332991 is engaging with cdk4/6 to inhibit phosphorylation of Rb at Ser780 and decrease proliferation in the tumors. Interestingly, while inhibition of cdk4/6 would be expected to have a cytostatic effect, several lines of evidence point towards a cytoreductive effect in vivo. The results of this study strongly support use of PD0332991 in a clinical trial for pediatric ATRT and medulloblastoma patients.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0152. DRUGGING MYCN THROUGH AURORA KINASE A TO TARGET MEDULLOBLASTOMA

W Clay Gustafson 1, Justin Meyerowitz 1, Erin Nekritz 1, Elise Charron 1, Katherine Matthay 1, Nicholas Hertz 1, Kevan Shokat 1, William Weiss 1

Abstract

INTRODUCTION: Amplification or mis-expression of MYC proteins contributes to a range of pediatric brain cancers including most subtypes of medulloblastoma. MYC proteins are tightly regulated at the level of protein stability. Stabilization of MYCN protein in particular proceeds through a kinase-independent function of the mitotic kinase, Aurora Kinase A, with expression of Aurora Kinase A itself reported to correlate negatively with survival in medulloblastoma (Neben et al, Cancer Research 2004). METHOD: An array of MYCN-driven cancer cell lines were acquired through the UCSF Cell Culture Core. Lentiviral and bacterial constructs were generated to express MYCN and mutants were generated by PCR-based site-directed mutagenesis (Stratagene). Aurora A was purified from overexpressing bacteria, crystals were generated by hanging drop vapor diffusion. Standard chemical synthesis and crystallography were performed. Western blots and in vitro Aurora A kinase assay were performed by standard techniques using antibodies to MYCN (Abcam), Aurora A, Histone H3, phospho-Histone H3 (all from Cell Signaling), and GAPDH (millipore). RESULTS: We synthesized and screened a novel class of inhibitors to identify agents that finessed degradation of MYCN protein across several MYCN-expressing cell lines. These structures demonstrate that a bulky chemical moiety in CD532 (our lead compound) clashes sterically with structures in the amino terminus of Aurora Kinase A, resulting in a hinge-like movement of the amino-terminal lobe away from the carboxy terminal lobe of Aurora A. In comparing structures of free Aurora Kinase, Aurora Kinase bound to clinical inhibitors, and Aurora Kinase bound to CD532, the degree of hinge movement correlated directly with degradation of MYCN. CONCLUSIONS: These structural data,coupled with mechanistic studies and biochemical assays, show potent MYCN protein degradation above the effects of simple nanomolar level inhibition of Aurora Kinase A kinase activity. This class of inhibitors, which disrupts stabilizing interactions between Aurora A and MYCN, represents new candidate agents to target medulloblastoma and other MYC-driven cancers.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0154. A HUMAN NEURAL STEM CELL MODEL OF MYC-DRIVEN MEDULLOBLASTOMA REVEALS GLUTAMINE METABOLISM AS A POTENTIAL THERAPEUTIC TARGET

Allison Hanaford 1, Eric Raabe 1, Charles Eberhart 1

Abstract

INTRODUCTION: Medulloblastoma comprises four subgroups with different associated mutations and clinical prognoses. Group 3 tumors have increased MYC levels and the worst clinical prognosis. MYC over-expression in cancer can lead to increased glutamine metabolism and glutamine addiction. We hypothesized that human neural stem cells derived from the developing cerebellum transduced with MYC and cooperating oncogenes would create a genetically accurate model of group 3 medulloblastoma for pre-clinical testing. We further hypothesized that MYC-driven medulloblastoma would exhibit increased glutamine metabolism and be sensitive to inhibitors of glutamine metabolism. METHOD: We transduced cerebellar derived human neural stem cells with lentivirus coding for c-MYC, dominant negative R248W p53, constitutively active AKT, and hTERT. We also created neurospheres expressing subsets of these constructs as controls. We measured proliferation in vitro with BrdU and MTS assays and generated orthotopic xenografts using standard techniques. Tumors were processed for histology and stained by immunohistochemistry for markers of neuronal and glial differentiation. After treatment with the glutamine anti-metabolite (2S)-Amino[(5S)-3-chloro-4,5-dihydro-1,2-oxazol-5-yl]ethanoic acid (acivicin), we determined apoptosis by flow cytometry (cell cycle and annexin staining) and cleaved caspase 3 immunofluorescence. We determined glutaminase expression by western blotting. RESULTS: R248Wp53/MYC/AKT/hTERT transformed cerebellar stem cells formed aggressive orthotopic xenograft tumors with large cell/anaplastic histology and spinal metastases. Control MYC-alone and R248Wp53/MYC/hTERT transduced cells formed tumors with increased latency and decreased penetrance. MYC expression positively correlated with increased expression of glutaminase. Acivicin decreased cell proliferation in MYC-containing neurospheres compared to those without MYC (p = 0.012). Acivicin treatment of MYC expressing cells led to a 65% increase in apoptosis compared to MYC-negative cells. Forced MYC expression in the low-MYC UW228 medulloblastoma cell line also led to increased glutaminase expression and increased sensitivity to acivicin (p = 0.03). CONCLUSIONS: Human cerebellar stem cells can be transformed by c-MYC and cooperating oncogenes, resulting in a models that recapitulates group 3 medulloblastoma histologically and clinically. These cells and classical high-MYC medulloblastoma cell lines are sensitive to glutamine anti-metabolites, while non-MYC driven medulloblastoma and SV40 immortalized human cerebellar stem cells are resistant. Our group 3 human neural stem cell medulloblastoma model data suggest that glutamine metabolism may be a therapeutic target in MYC-driven medulloblastoma, and that glutamine anti-metabolites may be useful clinical agents.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0155. RATIONALE FOR TRASTUZUMAB THERAPEUTIC ANTIBODY TREATMENT IN EPENDYMOMA

Andrea Griesinger 1, Andrew Donson 1, Lindsey Hoffman 2, Vladimir Amani 1, Diane Birks 1, Michael Handler 2, Jean Mulcahy-Levy 2, Nicholas Foreman 2

Abstract

INTRODUCTION: Ependymoma (EPN) currently experience 50% mortality at 10 years. Standard therapy consisting of surgery and radiation has not been significantly improved by the addition of chemotherapy. New therapeutic approaches are therefore necessary to improve the outcome for these patients. We have previously reported that the amount of tumor-infiltrating microglia/macrophage (M/M) in EPN at diagnosis is positively correlated with survival. We therefore hypothesize that immunotherapy will be effective in the treatment of EPN. Based on the success of Phase III clinical trials of therapeutic antibodies in neuroblastoma we explored the rationale for such an approach in EPN. METHOD: Global gene expression microarray analysis data (Affymetrix U133plus2) was used to identify EPN specific tumor-associated antigens that are targetable by existing therapeutic antibodies. Targeting of EPN cells by trastuzumab and potential for Fc-receptor mediated-antibody dependent cell-mediated cytotoxicity (ADCC) was validated in primary patient tumor samples using flow cytometric analysis of disaggregated tumor samples. Ex-vivo primary cultures established from disaggregated EPN were used to test the effect of treatments on immune infiltrates and tumor viability. RESULTS: HER2, targetable by trastuzumab/Herceptin, was shown to be overexpressed in EPN versus normal brain and other pediatric and adult brain tumor types by microarray analysis. Flow cytometric analysis showed that fluorescently-labeled trastuzumab was selectively bound to HER2 expressing EPN tumor cells and M/M cells. Further flow cytometric studies demonstrated that EPN infiltrating M/M express high levels of high-affinity Fc-gamma receptor CD64. Using ex-vivo primary cultures established from dissagregated EPN, the M/M population could be expanded with a subsequent reduction of viable tumor cells when treated with GM-CSF. Further studies are showing promising results with combination treatment of trastuzumab and GM-CSF. CONCLUSIONS: Together these results provide strong rationale for the use of trastuzumab in combination with immune stimulant GM-CSF in EPN.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0156. BRAF STATUS IN PERSONALIZING TREATMENT APPROACHES FOR PEDIATRIC GLIOMAS

Aleksandra K Olow 1, Tina Dasgupta 1, Xiaodong Yang 1, Sabine Mueller 2, Rintaro Hashizume 3, Ilan Kolkowitz 2, William Weiss 2, Adam C Resnick 5, Angela J Sievert 5, Theodore Nicolaides 4, Michael D Prados 3, Mitchel S Berger 3, Nalin Gupta 3, C David James 3, Daphne A Haas-Kogan 1

Abstract

INTRODUCTION: Pediatric low-grade gliomas (PLGGs) represent an excellent platform for personalized approaches to cancer treatment. Activation of mitogen protein kinase (MAPK) and phosphatidylinositol-3' kinase (PI3K) in PLGGs suggest the potential efficacy of agents that target these key growth regulatory pathways. BRAF, a kinase within the MAPK pathway, is activated by missense mutation (V600E) in ∼20% of grade 2-4 pediatric gliomas. In addition to RAS/MEK/MAPK/ERK, the PI3K/AKT/mTOR signaling cascade is activated in ∼half of PLGGs. In the current study, we asked whether knowledge of individual tumor BRAF genotype can guide selection of the best combination of signaling inhibitors for individual pediatric gliomas. METHOD: We used glioma cell lines containing BRAFV600E (AM38) or wild type BRAF (SF188, SF8628), and normal human astrocytes immortalized with hTERT and E6/E7 expressing retrovirus. Signaling inhibitors used were everolimus (RAD001, mTOR inhibitor, 4nM), PLX4720 (BRAFV600E specific inhibitor, 5 microM), AZD6244 and GDC-0973 (MEK inhibitors, 1 microM), and 0.05% DMSO as control. Cell cycle distribution was determined 24 hours after treatment using flow cytometry to quantitate BrdU and 7-AAD staining according to FITC BrdU Flow Kit (BD). Apoptosis was assessed 72 hours after treatment by flow cytometry quantitation of Annexin V and by PI positive cells. RESULTS: AM38 cells (BRAFV600E) showed significantly increased apoptosis, diminished S phase, and G1 cell cycle arrest after single agent everolimus, PLX4720, AZD6244, or GDC-0973. Combinations of PLX4720 and everolimus, PLX4720 and AZD6244, or PLX4720 and GDC-0973 were equally effective in enhancing effects on apoptosis and cell cycle arrest in BRAFV600E cells, compared to single agents. Pediatric glioma lines SF188 and SF8628 (BRAF WT) were also sensitive to everolimus, AZD6244 and GDC-0973, but not PLX4720. Combination of everolimus and AZD6244 or everolimus and GDC-0973 was superior to single agent treatments. No single agent or combination affected apoptosis or cell cycle in NHA. CONCLUSIONS: The use of BRAF status to determine effective combination therapies is an important consideration for personalized treatment of PLGGs. For BRAFV600E tumors, combination of PLX4720 and everolimus or PLX4720 and MEK inhibitor are equally efficacious and superior to single agent treatment, but we consider the former combination may be better tolerated for long-term treatment of children with PLGGs. For BRAF WT PLGGs, combination of everolimus with MEK inhibitor is superior to single agent therapies and would represent our treatment of choice for PLGGs with this genotype in future clinical trials. We next focus on optimal treatment of KIAA1549:BRAF-expressing PLGGs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0157. NOVEL ROLE FOR ENHANCING IMMUNOTHERAPY AGAINST PEDIATRIC BRAIN TUMORS USING HEMATOPOIETIC STEM CELLS

Catherine Flores 1, Christina Pham 1, David Snyder 1, Luis Sanchez-Perez 1, Darell Bigner 1, John Sampson 1, Duane Mitchell 1

Abstract

INTRODUCTION: Adoptive T-cell therapy is an effective treatment modality for solid tumors, leading to objective clinical responses, including >40% durable complete response rate of metastatic lesions within the CNS. We have pioneered a platform of adoptive cellular therapy (ACT) employing total tumor RNA-pulsed dendritic cells to expand polyclonal tumor-reactive T-lymphocytes in vitro. The anti-tumor efficacy of adoptively transferred T-cells is significantly enhanced under myeloablative host conditioning and hematopoietic stem cell rescue, leading to significant survival benefit and cures. The role of HSCs in immunotherapy is unexplored, but we demonstrate that HSC-T cell interactions in adoptive immunotherapy lead to enhanced anti-tumor efficacy. METHOD: Primary dendritic cells derived from murine bone marrow were pulsed with total RNA derived from a syngenic high-grade astrocytoma (KR158B-luc) and used to generate tumor-reactive T lymphocytes (TTRNA-T cells) ex vivo. TTRNA-T cells were adoptively transferred into intracranial tumor bearing mice following myeloablative host conditioning and HSC rescue. RESULTS: In a highly invasive murine astrocytoma model, ACT and HSC co-transfer, resulted in significant prolongation of median survival and cures of established tumors. These studies revealed greater T-cell infiltration of intracranial tumors in mice receiving MA + HSCs and long term T-cell persistence within the tumor microenvironment. Specific co-localization and persistence of tumor-reactive T-cells and HSCs were observed within the tumor microenvironment for >60 days post-transfer. Mechanistic studies demonstrated that HSCs significantly enhance intratumoral localization of T-cells via the MIP1α/CCR5 axis, showing a direct correlation between the presence of HSC-derived chemokines and increased numbers of tumor reactive T-cells at the tumor site. CONCLUSIONS: Our novel data reveals that HSC-T cell interactions within the tumor microenvironment modulate immunotherapy by guiding the intratumoral localization of T-cells, thereby playing a role in facilitating the eradication of infiltrative malignant gliomas.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0158. MALIGNANT TRANSFORMATION OF A DESMOPLASTIC INFANTILE GANGLIOGLIOMA - COMPARATIVE GENETIC HYBRIDIZATION ANALYSIS OF THE PRIMARY GLIOMA AND TRANSFORMED MALIGNANT GLIOBLASTOMA

Vikram Prakash 1, Jacqueline Batanian 1, Miguel Guzman 1, Thomas Geller 1

Abstract

INTRODUCTION: DIG (Desmoplastic Infantile Ganglioglioma) is a rare intracranial neoplasm classified as WHO grade I tumor under neuronal and mixed neuronal glial tumors (under 2007 World Health Organization brain tumor classification,). It is usually considered to have a good prognosis but 40 % of the cases require additional medical, radiation and/or further surgical intervention and 15% of infants and children develop leptomeningeal spread or die from DIG(1). Transformation into malignant glioblastoma has been described in literature but no studies have been conducted to detect the genetic alterations associated with the transformation. METHOD: A DIG tumor in a 2 mo old boy that showed aggressive behavior, requiring debulking initially at 2.5 mo age and subsequently at 10 mo age following tumor progression, was treated with chemotherapy, and subsequently by observation. At 8.5 yrs age the patient presented with recurrent seizures in association with malignant transformation into glioblastoma. Chromosome microarray analysis (CMA) using oligo array was performed on the biopsy specimen obtained at 2 mo age and on the subsequent transformed malignant glioblastoma. RESULTS: Following the clinically stable course of 7.5 years duration at glioblastoma transformation additional debulking and chemotherapy was employed. One year subsequently, the child remains stable with moderate neurological deficits. CMA using oligo array performed on the biopsy specimen obtained at 2 mo age did not show any significant abnormality, however there were significant genomic deletions and duplications associated with the glioblastoma from the same tumor subsequently. These included multiple genomic losses (4q, loss of chromosome Y, deletion of 6q) and a gain of 5q. Amplification of 12q14 involving the genes GL1 and CDK4 was identified. CONCLUSIONS: The complexity of cytogenetic abnormalities is suggestive of genomic instability and indicative of an aggressive tumor progression. Deletion of 4q, the loss of chromosome Y, and amplification of GLI and CDK4 genes are associated with pediatric glioblastomas; (CDK4 is part of Rb/p16INK4a/cyclin-D/CDK4 pathway also associated with malignant transformation of low grade ganglioglioma); and deletion of 6q and gains in 5q are frequently found in pediatric malignant astrocytomas. Our case illustrates the rare recurrence of DIG with malignant transformation into glioblastoma and its associated genetic alterations.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0159. DEVELOPMENTALLY REGULATED ANTIGENS FOR IMMUNOLOGIC TARGETING OF MEDULLOBLASTOMA SUBTYPES

Christina D Pham 1, Yanxin Pei 2, Catherine Flores 1, David Snyder 1, Darell D Bigner 1, John H Sampson 1, Robert J Wechsler-Reya 2, Duane A Mitchell 1

Abstract

INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Immunotherapy presents a promising alternative by specifically targeting cancer cells and to date, there have been no successful immunologic applications targeting MB. Using total embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHOD: We have created two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the role of the immune system in the specific targeting of different MB subtypes. To assess the capacity of fetal antigens to serve as immunologic targets for MB, T cells specific to the total RNA contents of fetal mouse cerebellum (P5) were generated and tested for their reactivity to target cells pulsed with total RNA from two MB subtypes and normal brain. Immune responses were also evaluated by measuring cytokine secretion following re-stimulation of activated T cells with MB tumor cell targets. RESULTS: Our studies have demonstrated the capacity to generate T cells specific to the developing cerebellum that recognize and respond to surrogate target cells expressing the same cohort of antigens in vitro, confirming the immunogenicity of developmentally regulated antigens. Additionally, we have shown that developmental antigen-specific T cells recognize tumor cells of two immunologically distinct subtypes of MB and have no cross-reactivity with the normal cerebellum. CONCLUSIONS: Our preliminary data suggest that the use of developmental antigens can safely target multiple MB subtypes with equal effectiveness compared to previously established total tumor strategies and can be done without potential toxic off-target effects to the surrounding brain. The use of developmental antigens as specific, yet safe immunologic targets in the treatment of different MB subtypes remains to be thoroughly tested in its ability to mediate tumor cell killing in vivo in our lab's preclinical MB murine models.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0160. Bmi1 REGULATES GLI ACETYLATION THROUGH CULLIN3 IN MEDULLOBLASTOMA STEM CELLS

Randy Van Ommeren 1, Chitra Venugopal 1, Branavan Manoranjan 1, Nicole McFarlane 1, Robin Hallett 1, John Hassell 2, Sandra Dunn 1, Sheila Singh 1

Abstract

INTRODUCTION: Constitutive activation of the Shh pathway in neural progenitor cells of the external granular layer of the cerebellum is a frequent driver of brain tumour initiating cells (BTICs) in medulloblastoma (MB). Recently, we have shown that activation of the Shh pathway leads to an increase in production of Bmi1, a polycomb repressor which promotes ‘stemness’ in BTICs. Our current work demonstrates that up-regulation of Bmi1 by the Shh pathway leads to repression of cullin3 transcription. Cullin3 has been shown to ubiquitinate HDAC1, leading to increased acetylation of the Gli transcription factors which can further activate the Shh pathway. METHOD: We use MB BTICs from cell lines and primary tumours, grown in neural stem cell media. Bmi1 and Cul3 expression is modulated using overexpression (OE) and lentiviral-mediated shRNA knockdown (KD) vectors. Subsequently, mRNA and protein levels of downstream genes are assessed by RT-PCR and Western blotting (WB). Gli acetylation is assessed by immunoprecipitation using anti-acetyl lysine antibody on WB. Chromatin immunoprecipitation (ChIP) using Bmi1 antibody on CD15+ cells and subsequent PCR amplification of Cul3 promoter regions will elucidate Bmi1 occupancy of Cul3 promoter. Functional significance of Cul3 knockdown in BTICs will be assessed in vivo, using a NODSCID xenograft model. RESULTS: To date, ChIP analysis has demonstrated that Bmi1 is enriched at the Cul3 promoter in CD15+ stem/ progenitor cells though not in CD15- non-stem cells. Overexpression of Bmi1 has been shown by RT-PCR to cause reduced levels of Cul3 transcript, and an inverse result was observed for Bmi1 knockdown. Further, western blot analysis has found that overexpression of Bmi1 leads to a reduction of Cul3 protein and a corresponding increase of HDAC1 in Med8a tumor spheres. CONCLUSIONS: Our data suggests the existence of a positive feedback loop whereby Bmi1 can repress transcription of the Cul3 E3 ligase. This in turn leads to reduced ubiquitination of HDAC1 and increased Gli deacetylation. Deacetylated Glis further activate Bmi1 transcription, driving the positive feedback system. We have previously demonstrated MB BTICs to be Shh-receptive cells that respond to ligand produced by bulk tumour. Our new findings suggest a novel mechanism whereby exposure to the ligand might lead to aggressive activation of the Shh pathway and produce a bona fide BTIC, providing a novel set of drug targets for future therapies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0161. SURVIVAL ADVANTAGE WITH EVEROLIMUS (RAD001) COMBINED WITH A SELECTIVE BRAFV600E INHIBITOR IN A XENOGRAFT MODEL OF BRAFV600E-MUTANT PEDIATRIC GLIOMA

Tina Dasgupta 1, Aleksandra Olow 1, Xiaodong Yang 1, Rintaro Hashizume 1, Sabine Mueller 1, Theodore Nicolaides 1, Ilan Kolkowitz 1, William Weiss 1, Michael Prados 1, Nalin Gupta 1, C David James 1, Daphne Haas-Kogan 1

Abstract

INTRODUCTION: Pediatric gliomas commonly recur after resection, and, as a result, these tumors are frequently treated with adjuvant chemotherapy, or radiation. We have previously shown that the PI3K/Akt/mTOR pathway is activated in pediatric brain tumors, and this activation confers tumor sensitivity to mTOR inhibition. Similarly, we have demonstrated that tumors with activating BRAFV600E mutation, that occur in as much as 20% of pediatric grade II-IV gliomas, are responsive to V600E-targeted chemotherapy. In the current study, we have investigated the efficacy of mTOR + BRAFV600E combination therapy, using in vitro and in vivo approaches. METHOD: BRAFV600E-targeted inhibitor PLX4720 and mTOR inhibitor everolimus (RAD001) were used. In vitro cell proliferation and apoptotic response of inhibitor treatments were examined using clonogenic assays and flow cytometry. In vivo, mice with flank xenografts, established with a BRAFV600E mutant pediatric low-grade glioma (BT40), were randomized to receive vehicle only (control), monotherapy, or a combination of PLX4720 + everolimus. Animals were euthanized per protocol, and followed for survival and response to treatment by monitoring tumor volume, and by immunohistochemical analysis of tumor tissues for proliferation (Ki-67) and apoptotic response indicators. RESULTS: Clonogenic assay results showed synergistic activity between PLX4720 and everolimus for BRAFV600E gliomas. Flow cytometry revealed increased G1 arrest in BRAFV600E cells treated with combined inhibitors, in relation to treatment with monotherapy. Cells with wild-type (WT) BRAF were nonresponsive to PLX4720, and combination therapy did not improve upon the anti-tumor effects of everolimus monotherapy for BRAFWT cells. In the in vivo model of BRAFV600E glioma, combined treatment with PLX4720 + everolimus led to a statistically significant survival advantage in relation to either monotherapy. In vivo and in vitro results indicated increased apoptosis and DNA damage in BRAFV600E mutant cells from combination therapy. CONCLUSIONS: To our knowledge, this is the first in vivo demonstration of combinatorial activity of a BRAFV600E (PLX4720) and mTOR inhibitor (everolimus), as applied to potential glioma treatment. Our results suggest that apoptosis, DNA damage and cell cycle arrest are enhanced through use of this inhibitor combination, and their combined use extends animal subject survival. Because our in vivo studies were conducted using xenografts derived from pediatric gliomas, and because both the clinical analogue of PLX4720 (vemurafenib) and everolimus are FDA-approved to treat other cancers, these results have direct implications for clinical trial design in children with BRAFV600E mutant brain tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0162. DEVELOPMENT AND ANALYSIS OF A GENETIC MODEL OF CHOROID PLEXUS TUMOR

Haotian Zhao 1, Li Li 1, Katie Picotte 1

Abstract

INTRODUCTION: Choroid plexus tumors are intraventricular papillary neoplasms arising from choroid plexus epithelium. They occur most often in childhood and comprise 10–20% of all brain tumors in infants. Choroid plexus papilloma is a benign tumor, whereas choroid plexus carcinoma is malignant and most commonly found in pediatric population. Despite good prognosis for choroid plexus papilloma after complete surgical removal, incompletely resected or inaccessible papillomas or choroid plexus carcinomas are often associated with poor outcomes. Previous studies implicate Notch signaling in choroid plexus tumorigenesis. In this study, we developed and characterized a genetic model of choroid tumors based on Notch pathway activation. METHOD: Math1-Cre trasgneic strain was bred with Rosa26-NICD1 strain that express Notch1 intracellular domain (NICD1) in a Cre-dependent manner. Math1-Cre/Rosa26-NICD1 animals were further crossed to GFP reporter strain to label Math1-positive lineage from rhombic lip. Tumor cell proliferation, differentiation and survival were analyzed by immunohistochemistry and immunoblotting analysis. Gene expression was examined by quantitative RT-PCR analysis and immunostaining. RESULTS: Math1-postive rhombic lip lineage was detected in a small fraction of choroid plexus epithelium of 4th ventricle. A hyperplasia of in the choroid plexus epithelium derived from Math1-positive rhombic lip was observed in Math1-Cre/Rosa26-NICD1 animals. Morphological analysis revealed papillary and intraventricular growth of epithelial cells that exhibited enhanced proliferation compared to control animals in early postnatal period. Tumor cells expressed Lmx1a, Otx2 and Aquaporin 1, markers for choroid plexus epithelial cells. In addition, tumor cells express acetylated tubulin, suggesting the presence of cilia structures. Gene expression analysis demonstrated that tumor cells express NICD1 and exhibit constitutively active Notch signaling. CONCLUSIONS: Math1-positive rhombic lip lineage contributes to choroid plexus epithelium and is sensitive to Notch1-driven tumor formation. We have developed a genetic model of choroid plexus tumor that exhibits characteristics similar to pediatric choroid plexus tumors. We will continue to examine mechanisms and signaling pathways that drive tumor cell proliferation and malignant transformation in this model. Study of choroid tumors in this novel model will bring crucial insights into the mechanisms driving tumor formation and new directions for therapeutic development.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0163. ESTABLISHING ZEBRAFISH MODELS OF CNS PRIMITIVE NEUROECTODERMAL TUMORS

Rodney Stewart 1, Kate Modzelewska 1, Elena Boer 1, Daniel Picard 2, Annie Huang 2

Abstract

INTRODUCTION: Primitive Neuroectodermal Tumors (PNETs) of the CNS are rare, aggressive childhood tumors whose molecular and cellular origins are not known. Recent integrative genomic approaches on a large collection of hemispheric PNETs have revealed at least 3 distinct PNET subtypes: Primitive-neuronal, Oligoneural and Mesenchymal. These studies suggest that distinct cellular origins and oncogenic events drive the formation of different PNET subtypes. By modeling these specific changes in the neuroectoderm of the zebrafish we have generated a novel vertebrate CNS tumor model that closely resembles the human oligoneural PNET. METHOD: To model CNS PNETs we analyzed published gene expression databases to identify potential oncogenic events and markers for cell of origin. For example, oligoneural PNET subtype tumors have elevated expression of SOX10 and OLIG2, suggesting a glial precursor origin. In addition, NRAS expression and p53 pathway mutations are associated with PNET tumorigenesis. To target these changes in sox10-expressing progenitor cells we generated transgenic lines that express fluorescently tagged human NRAS under the control of the sox10 promoter in a p53 mutant background. CNS tumor onset was monitored by mCherry fluorescence and characterized at histopathological and molecular levels. RESULTS: Tg(Sox10:mCherry:NRAS); p53 animals develop CNS tumors by 8 weeks of age with 50% penetrance. Tumors grow aggressively throughout the CNS and surrounding tissues, with animals succumbing to disease by 38 weeks. Pathological analysis show small round blue cell tumor histology with IHC consistent with human PNET tumors (synaptophysin, GFAP, Nestin). Differential gene expression analysis based on RNA-Seq from 8 different fish PNET tumors and controls shows activation of genes involved in midbrain and glial development. Cross-species comparisons of PNET gene expression signatures reveal highly conserved genetic programs drive oligoneural PNET formation. CONCLUSIONS: This is the first zebrafish model of a human brain tumor and the first model of the oligoneural PNET subtype. These models can be used to identify conserved molecular mechanisms involved in PNET formation and invasion. We are also generating inducible PNET models using CRE/LOX approaches that will allow us to produce thousands of these tumor animals at low cost for future in vivo drug screens.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0164. PLK1 INHIBITION LEADS TO INACTIVATION OF THE RSK/YB-1 PATHWAY AND MAY SERVE AS A SURROGATE FOR SMALL MOLECULES IN CLINICAL TRIALS

Daniel Radiloff 1, Cathy Lee 1, Sandra Dunn 1

Abstract

INTRODUCTION: Polo-like kinase-1 (PLK1) is essential for the sustained growth of brain tumors. Our laboratory previously published that PLK1 inhibition with either siRNA or the small molecule BI-2536 suppresses the growth of glioblastoma multiforme (GBM) and medulloblastoma (MB) in vitro and in orthotopic mouse models. More recently we determined that PLK1 is associated with relapse and poor survival in pediatric MB patients. The underlying molecular mechanisms of PLK1's role in promoting tumor growth and drug resistance remained largely undefined. Moreover, a robust surrogate marker(s) for PLK1 inhibition is currently lacking in clinical trials. METHOD: Gain of function and loss of function approaches were used to examine PLK1's downstream signaling activities. Human recombinant PLK1 was transfected into HEK293 cells and signaling changes were assessed by immunoblotting. To examine downstream PLK1-regulated signaling events in GBM, it was silenced with siRNA into SF188 pediatric brain tumor cells. We utilized antibody arrays to assess the impact of PLK1 inhibition on the proteome. The second generation PLK1 pharmacological inhibitor BI-6727 was also used to inhibit PLK1 in brain tumor cells lines to address changes in signaling and cell growth. RESULTS: In HEK293 cells, transfection of hPLK1 induced the activation of RSK/YB-1 pathway, which belongs to the MAPK signaling cascade. Using antibody arrays, the MAPK pathway was also implicated in PLK1 signaling. Inhibiting PLK1 with siRNA blocked P-RSK and P-YB-1. Likewise, BI-6727 inhibited this pathway in a dose-dependent manner. In primary pilocytic astrocytoma cells PLK1 was expressed at low levels. Therefore we are transfecting PLK1 into the cells to activate the RSK/YB-1 pathway. Cell growth and response to therapy will be addressed. Conversely, PLK1 will be inhibited in primary GBM and the effect on the pathway will be reported. CONCLUSIONS: There are several PLK1 inhibitors in clinical trials for the treatment of adult cancers, however, they have not been evaluated in children with brain tumors. One of the challenges is discerning whether or not the inhibitors are active and this would be achieved using surrogate markers that rely on PLK1 signaling. We conclude that the RSK/YB-1 pathway could potentially serve as a surrogate for PLK1 inhibitors in the clinic.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0165. TARGETING THE NOTCH AND mTOR PATHWAYS IN DIFFUSE INTRINSIC PONTINE GLIOMA

Marianne Hütt 1, Javad Nazarian 2, Antoinette Price 1, Kah Jing Lim 1, Katherine Warren 3, Howard Chang 4, Charles G Eberhart 1, Eric H Raabe 1

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor known to have frequent alteration of mTOR signaling. We have established JHH DIPG1, a human DIPG xenograft and neurosphere cell line from a rapid autopsy specimen obtained through the Mid Atlantic DIPG Consortium. This DIPG cell line expresses high level phospho-S6 and phospho-AKT(473), indicating activation of TORC1 and TORC2. Notch 1 and 3 and Notch downstream effectors, Hes1, Hes5 and Hey1, are also highly expressed in JHH DIPG1 and a second DIPG line, SU DIPG1. We targeted Notch and mTOR pathways using gamma-secretase (GSI) and TORC inhibitors. METHOD: Real-time PCR was used to investigate levels of Notch downstream targets in DIPG cell lines. TORC1 and TORC2 levels were assessed by investigating expression of phospho-S6 and phospho-AKT(473) on Western Blot. Cell proliferation was assessed using the bromodeoxyuridine (BrdU) incorporation assay. RESULTS: Treatment of DIPG cell lines with a TORC1 inhibitor attenuates cell proliferation by 60% in treated versus non-treated cells (p < 0.005). TORC1 inhibition causes a dose-dependent phospho-S6 reduction, with concomitant upregulation of phospho-AKT(473). Notch targeting using a GSI leads to a dose-dependent reduction in cell proliferation of up to 30% as well as inhibition of phospho-AKT(473) (p = 0.05). However, GSI in combination with TORC1 inhibition has only a modest effect on phospho-AKT(473). TORC-kinase inhibition using PP242 reduces the levels of phospho-AKT(473) as well as phospho-S6. CONCLUSIONS: Targeting TORC1 decreases DIPG cell proliferation while increasing TORC2 levels. Inhibition of NOTCH targets TORC2 complexes and negatively affects cell proliferation. Simultaneous use of TORC1 inhibitor and GSI suppresses both complexes; however the increase in TORC2 by TORC1 inhibition persists. Because TORC1 and TORC2 inhibition individually decrease cell proliferation, simultaneous suppression of TORC1 and TORC2, may prove to be synergistic in DIPG. Simultaneous TORC1 and TORC2 inhibition was achieved using a TORC-kinase inhibitor. We are currently exploring the phenotypic effects of dual-TORC1/2 inhibition and the role of the Notch and mTOR pathways in DIPG in murine xenograft studies.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0166. MODELING PEDIATRIC GLIOMA SUBGROUPS IN MICE AND HUMANS

Anders Persson 1, Miller Huang 1, Devin Chandler-Militello 2, Nan Li 1, Mitchel Berger 1, David James 1, Steve Goldman 2, William Weiss 1

Abstract

INTRODUCTION: Transcriptomal subgroups of childhood GBMs are associated with IDH1 (R132H) mutations, H3F3A (K27/G34) mutations, and PDGFRA amplifications. Most pediatric GBMs (except PDGFRA amplified tumors) displayed mutations in the p53 gene. The location and age at diagnosis suggest that GBM subgroups arise from different precursor cells. We and others have previously shown that the proneural tumor oligodendroglioma can arise from oligodendrocyte progenitor cells (OPCs). We have validated data from other groups showing that astrocytomas can arise from neural stem cells (NSCs). Developments of pediatric gliomas have traditionally been modeled using mouse models rather than human precursor cells. METHOD: We use the RCAS/tv-a system to drive expression of PDGF-BB, R132H mutation, K27 mutation, and G34 mutation. We infect OPCs and NSCs through injections into supratentorial and brain stem regions at early postnatal ages in mice displaying p53 mutations. Isolation of precursors from different regions followed by infection of tv-a expressing cells allow us to study effects on differentiation, self-renewal, and proliferation. Gene expression and methylation of infected cells are characterized. Lentiviral infection of cultured human fetal OPCs and NSCs with PDGFB, R132H mutation, K27 mutation, and G34 mutation in combination with shRNA against p53 is used to induce transformation. RESULTS: Stereotactic injections demonstrated that OLIG2-expressing OPCs represent a target for transformation in both mouse and human cells. OLIG2-tva:p53 mutant mice infected with RCAS-PDGF-BB developed high-grade gliomas after 2-4 months. Infection of OPCs in vitro using RCAS-PDGF-BB virus resulted in a rapid expansion along with a block in differentiation. Tumors generated by other oncogenic drivers are currently being evaluated. CONCLUSIONS: As the most widely expressed cycling cell population, OPCs is a likely origin for different childhood gliomas. Our studies also suggest that a block in differentiation is an early event in gliomagenesis. Transformation of human precursor cells in vitro followed by xenografting into mice represent a model to study sequential events during gliomagenesis.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0167. PROLIFERATIVE PROGENITOR CELLS OF THE POSTNATAL PONS: IMPLICATIONS FOR PONTINE GLIOMAGENESIS

Robert Lindquist 1, Matthew Tate 1, David Rowitch 1, Arturo Alvarez-Buylla 1

Abstract

INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is a fatal pediatric brain tumor of unknown developmental origin. The present study describes the postnatal growth of human pons and the proliferative progenitor cells of the postnatal human and mouse pons, providing a developmental context in which to identify candidate cells of origin for DIPG. METHOD: We first measured growth of human brainstem by MRI analysis of normal children. We then characterized proliferation and lineage analysis by immunohistochemistry in pediatric human post-mortem samples and in mouse brainstem, co-staining proliferative cells for various markers of astrocyte or oligodendrocyte lineage progression. RESULTS: We observed that proliferation levels in postnatal human pons are greatest in neonates and decline through infancy; cells of the basis divided more robustly than tegmentum. This correlates with MRI data that show the pons to grow fastest in the first months of life, and faster in basis than tegmentum, then slowing its growth in childhood (despite minimal proliferation after 6 months). Proliferation in postnatal mouse pons is also restricted to childhood, peaking shortly after birth, and is greater in basis than tegmentum. The mouse pons contains several anatomically and molecularly distinct proliferative progenitor populations that decline at different ages. CONCLUSIONS: The early postnatal pons contains abundant proliferative progenitor cells, particularly in the ventral pons, which may account for extremely rapid regional growth during the first months of life. Ongoing work will identify the lineage association of proliferative cells in the pons and whether targeting these cells is sufficient for de novo gliomagenesis.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0168. UNIQUE PATTERNS OF PROGRESSION IN RECURRENT PEDIATRIC EPENDYMOMA

Lindsey Hoffman 1, Andrew Donson 1, Diane Birks 1, Andrea Griesinger 1, Vladimir Amani 1, Michael Handler 1, Nicholas Foreman 1

Abstract

INTRODUCTION: Ependymoma (EPN) is the 3rd most common brain tumor in children. Despite standard therapy, most children with EPN relapse and ultimately die of their disease. Numerous molecular characteristics of newly diagnosed EPN have been elucidated, but the biology of relapsed EPN is largely unexplored. METHOD: We used transcriptome microarray (Affymetrix U133plus2) to analyze 13 matched pairs (26 total samples) of newly diagnosed and relapsed pediatric EPN. Normalization and unbiased hierarchical clustering were performed using Bioconductor R, and ontology analyses were performed using DAVID. RESULTS: Clustering confirmed previously reported posterior fossa sub-groups (Groups A and B) and reveals unique progression patterns. Tumors initially presenting as Group A (n = 8) change significantly at recurrence, evidenced by progression of three recurrences to Group B (1096 differentially expressed genes (p<0.05; fold>1.5). Conversely in Groups B, recurrent tumors cluster side-by-side with their primary (n = 5), implying transcriptomic similarity (347 differentially expressed). Ontology (actin cytoskeleton) and specific gene (NELLS2, LAMA2) analyses of differentially expressed genes support progression from Group A to Group B phenotype at recurrence, and enhancement of group B signatures at recurrence in those initially presenting as Group B. CONCLUSIONS: We are the first to report sub-group re-designation of recurrent pediatric EPN. We note a unique pattern of progression between sub-groups in which Group A tumors exhibit more transcritpomic change and become more similar to Group B tumors at recurrence. This suggests that therapy at recurrence should be tailored to the Group B phenotype.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0169. REFINED RADIOLOGICAL RESPONSE CRITERIA FOR EVALUATING LEPTOMENINGEAL METASTASIS - LM RESPONSE SYSTEM

Lisethe Meijer 1, David Walker 1, Richard Grundy 1, Sophie O'Dowd 2, Tim Jaspan 2, Rob Dineen 2

Abstract

INTRODUCTION: Response evaluation in leptomeningeal metastasis (LM) of CNS or solid tumours is based upon CSF histopathology, applicable for those with positive CSF, who are the minority. Radiological response systems (e.g. RANO, RECIST, McDonald, SIOP) do not include LM response criteria. We propose radiological criteria, applied to 5 anatomical regions, to evaluate LM response, integrating qualitative (linear or nodular disease) and quantitative (size/number of nodules and thickness/extent of surface enhancement) assessments, which can be used to describe LMM involvement and response. METHOD: Response system was defined in 5 anatomical areas: cerebrum/suprasellar cistern, ventricular lining, brainstem/cranial nerves, cerebellum, spinal canal/thecal sac. Quantitative scores (0 – 3) were defined as linear extent (<10, 10–15, >15 mm), linear thickness (<1, 1–5, >5 mm), nodular number (<3, 3–5, >5), nodular size (<5, 5–15, >15 mm). Total response per area defined as: complete response (CR), partial (PR): ≥50% reduction, stable disease (SD): <50% reduction/ < 25% increase, progressive (PD): ≥25% increase. Criteria were applied to 24 anonymized, paired scans from 12 patients, pre- and post-LM intrathecal (IT) treatment. Using T1 and 2 sequences pre- and post-contrast. RESULTS: Of 12 cases: 11 cerebral, 7 intra-ventricular, 7 brainstem, 9 cerebellar, and 6 spinal. Pretreatment CSF status assessed in 10: 6 negative, and 4 positive (2 cleared on treatment). Tumour response assessed regionally: cerebral: 5 SD, 4 PD, 2 CR, 1 no disease; ventricular: 3 SD, 1 PD, 3 CR, 5 no disease; brain stem: 3 SD, 3 PD, 1 PR, 5 no disease; cerebellar: 3 SD, 5 PD, 1 CR, 3 no disease; spinal: 3 SD, 2 PD, 1 CR, 4 no disease, 2 missing. Clinical assessments identified 2 who deteriorated, 6 improved, 4 missing. All but one died. CONCLUSIONS: These criteria permit detailed anatomical, qualitative and quantitative description of LMM, meeting the requirements for face validity. The system would meet the needs of future trials of IT/ intraventricular therapy directed at LM. It is superior to CSF assessment as it is informative for all patients. It should be evaluated with paired observers in larger cohorts to assess construct validity against anatomical region of drug delivery and clinical response.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0170. THE Y-BOX BINDING PROTEIN-1 CONVEYS TEMOZOLOMIDE RESISTANCE BY THE INHIBITING DNA DAMAGE RESPONSE PATHWAY THROUGH THE POLYCOMB REPRESSOR BMI-1

Abbas Fotovati 1, Daniel Radiloff 1, Nicole Coute 1, Joanna Triscott 1, James Chen 1, Stephen Yip 1, David Louis 1, Brian Toyota 1, Juliette Hukin 1, S Rod Rassekh 1, Ash Singhal 1, Chris Dunham 1, Sandra Dunn 1

Abstract

INTRODUCTION: The Y-box-binding protein-1 (YB-1) is an oncogenic transcription and translation factor that is highly expressed in adult and pediatric glioblastoma (GBM) but not in normal brain tissues. It is functionally activated by p90 ribosomal S6 kinase (RSK). We previously reported that YB-1 mediates resistance to temozolomide (TMZ) in GBM independent of MGMT promoter methylation status. However, the mechanism underpinning this is not understood. We hypothesized that YB-1 confers drug resistance through chromatin remodeling via BMI-1 given that we recently reported that they are strongly co-expressed in primary GBM. METHOD: YB-1 protein was assessed in tumors from GBM patients before and after TMZ treatment (n = 14 pairs) by immunohistochemistry. YB-1 localization was evaluated in drug refractory GBM cell lines (SF188 and BT74), using immunocytochemistry and in orthotopic xenografts by immunohistochemistry. To understand how YB-1 contributes to TMZ resistance, we used pRT-PCR arrays to profile gene expression changes in SF188 cells[P1]post-YB-1 and BMI-1 silencing. RSK was inhibited with siRNA or the small molecule inhibitor BI-D1870. Primary GBM isolates were cultured in monolayer and as neurospheres. RESULTS: YB-1 was expressed in all of the tumors before TMZ treatment and therapy did not reduce expression. TMZ-treated SF188 cells continue to proliferate with retaining YB-1 transcriptional activity. Silencing YB-1 or BMI-1 induced GADD45a, and RAD17 mRNA and protein, both involved in DNA damage recognition pathway. Treating SF188 cells with RSK1, RSK2 or RSK1/2 siRNA or BI-D1870 suppressed P-YB-1 and induced this pathway. RSK inhibition also inhibited their growth by 80%. YB-1 is highly activated in BT74 xenografts, which correlated with ki67. In vitro, BI-D1870 blocked BT74 growth 100%. BI-D1870 suppressed primary refractory GBM cell growth. CONCLUSIONS: Using an unbiased PCR array, we discovered that YB-1 silences GADD45a and RAD17, which are genes responsible for recognizing DNA damage. This occurs through the polycomb repressor BMI-1. The expression of these genes are reactivated by inhibiting the RSK/YB-1/BMI-1 pathway suggesting that this may be a new avenue to sensitize GBM cells to TMZ. In summary, YB-1 functions impose TMZ resistance by suppressing the expression of genes that recognize DNA damage, which prevents the GBM cells from undergoing apoptosis.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0171. MYC DRIVEN HUMAN NEUROSPHERE MODELS OF PRIMITIVE NEUROECTODERMAL TUMORS ARE SENSITIVE TO INHIBITORS OF GLUTAMINE METABOLISM

Sama Ahsan 1, Allison Hanaford 1, Isabella Taylor 1, Charles Eberhart 1, Eric Raabe 1

Abstract

INTRODUCTION: The MYC family of proteins promotes proliferation and an aggressive phenotype in diverse cancers. Primitive Neuroectodermal Tumors (PNETs) are high grade tumors that arise throughout the neuro- axis. One subset of PNETs is MYC amplified and some PNETs are known to express LIN28A, a key regulator of MYC. MYC expression in tumors leads to increased reliance on glutamine metabolism (i.e. Warburg effect).We hypothesize that MYC-driven PNET tumors will up regulate glutaminase and thus increase glutamine metabolism. We secondarily hypothesize that glutaminase inhibitors will selectively target MYC-driven PNET. METHOD: We used the MYC -positive PFSK cell line and a human neural stem and progenitor cell model of MYC-transformed PNET to test our hypothesis. Neurospheres derived from developing human cerebral cortex were transduced using lentivirus coding for c-MYC, dominant negative p53, constitutively active AKT, and hTERT. Controls were non-MYC immortalized cortex neurospheres. MYC and glutaminase expression were determined by western blot. The glutaminase inhibitors DON (6-Diazo-5-oxo-L-norleucine) and acivicin ((2S)-Amino[(5S)-3-chloro-4,5-dihydro-1,2-oxazol-5-yl]ethanoic acid) were used to disrupt glutamine metabolism. We measured cell proliferation using MTT assay and BrdU. We evaluated apoptosis using flow cell cycle analysis and cleaved caspase 3 immunofluorescence. RESULTS: R248Wp53/MYC/AKT/hTERT transformed cortex neurospheres expressed increased MYC and glutaminase compared to non-MYC driven neurosphere cells. R248Wp53/MYC/AKT/hTERT tranduced human neurospheres formed aggressive PNET-like tumors in orthotopic xenograft models. R248Wp53/MYC/AKT/hTERT transduced human neurospheres treated with DON showed a 48% reduction in BrdU incorporation compared to untreated cells (p < 0.01). DON and acivicin treatment of R248Wp53/MYC/AKT/hTERT neurospheres led to an increase in the apoptotic fraction of cells, as measured by flow cytometry (26% increase in apoptotic fraction with DON treatment vs. 44% increase with Acivicin treatment). In comparison, non-MYC driven human neurospheres did not show an increase in apoptosis with DON or acivicin treatment. CONCLUSIONS: MYC expression in conjunction with other oncogenic hits can transform human neural stem and progenitor cells, creating a model of aggressive PNET. This MYC-driven PNET model is susceptible to glutamine metabolic inhibitors, while cortical neurospheres immortalized with oncogenes other than MYC are resistant to these inhibitors. We are currently further investigating the mechanism of growth inhibition using BrdU analysis, cell cycle analysis, and cleaved caspase 3. Murine orthotopic xenograft experiments investigating the efficacy of DON and acivicin treatment in MYC-driven PNET are currently underway in our laboratory. These experiments suggest that glutamine metabolism may be a therapeutic target in PNET.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0172. GENETIC ABLATION OF THE bHLH TRANSCRIPTION FACTOR OLIG2 IS SYNTHETIC LETHAL TO BRAF ONCOGENES IN A MURINE MODEL OF PEDIATRIC LOW-GRADE ASTROCYTOMA

Yu Grace Sun 1, Charles Stiles 1

Abstract

INTRODUCTION: The p53 tumor suppressor mediates cellular responses (growth arrest and/or apoptosis) in response to ionizing radiation or genotoxic drugs. In previous studies, we have shown that the neurogenic transcription factor Olig2 opposes these p53-mediated responses to DNA-damaging agents in neurosphere cultures from high-grade adult gliomas. However, a second function p53 is to induce growth arrest or apoptosis in response to inappropriate mitogenic cues (i.e. oncogenes). We show here that this second function of p53 is also suppressed by Olig2. The findings are relevant to pediatric low-grade astrocytomas (PLGAs) characterized by activating mutations in BRAF. METHOD: A high percentage of juvenile pilocytic astrocytomas express the KIAA 1549 truncation fusion form of BRAF. Other PLGAs (e.g. gangliogliomas, diffuse astrocytomas, pleomorphic xanthoastrocytomas) express either the V600E point mutation of BRAF or the KIAA 1549 mutant. Notably these two versions of the BRAF oncogene are mutually exclusive in PLGAs. We used retroviral expression vectors to introduce these two oncogenic versions of BRAF into either neural progenitor cells isolated from wild type or Olig2 null mouse embryos. RESULTS: Neural progenitors from wild type mouse embryos (Olig2-positive) tolerated transformation with either form of the BRAF oncogene. However, both forms of the BRAF oncogene were toxic to Olig2 null progenitors. CONCLUSIONS: Genetic ablation of Olig2 in murine neural progenitors is synthetic lethal to transformation by oncogenic mutants of BRAF. The synthetic lethal relationship between Olig2 and BRAF oncogenes could have practical overtones for targeted therapy of BRAF-transformed PLGAs - the majority of which are wild type with respect to p53 status and are Olig2-positive.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0173. LncRNA PROfiLE OF GLIOBLASTOMA REVEALS THE POTENTIAL ROLE OF LNCRNAS IN CONTRIBUTING TO GLIOBLASTOMA PATHOGENESIS

Lei Han 1, Kailiang Zhang 1, Luyue Chen 1, Zhendong Shi 1, Peiyu Pu 1, Lun Dong 2, Chunsheng Kang 1

Abstract

INTRODUCTION: LncRNAs have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Our aim is to identify important lncRNAs that might play an important role in contributing to glioblastoma (GBM) pathogenesis by conducting lncRNA and mRNA profiles comparison between GBM and normal brain tissue. METHOD: The differentially expressed lncRNA and mRNA profiles of the tissue between GBM patient and age-matched donor without GBM diseases were analyzed via microarray. We proposed a novel model for the identification of lncRNA-mRNA targeting relationships that combined the potential targets of the differentially expressed lncRNAs with the differentially expressed mRNAs abundance data. Bioinformatic analysis of the predicted target genes (gene ontology, pathway and network analysis) were done for further research. RESULTS: The lncRNA microarray reveals differentially expressed lncRNAs between the GBM and normal brain tissues. In the GBM group, 654 lncRNAs were upregulated and 654 were downregulated (fold change ≥ 4.0 or ≤0.25, P < 0.01). We found 104 matched lncRNA-mRNA pairs for 91 differentially expressed lncRNAs and 84 differentially expressed genes. Target gene-related pathway analysis showed a significant change in PPAR pathways in the GBM group compared with the normal brain group (P < 0.05). By further conducting lncRNA gene network analysis, we found that the ASLNC22381 and ASLNC2081 were likely to play roles in the regulation of glioma signaling pathways. CONCLUSIONS: Our results indicated that lncRNAs expression profile in GBM tissue was significantly changed. These results may provide important insights into GBM mechanisms. This study also suggests that ASLNC22381 and ASLNC20819 might play important roles via their target IGF-1 in the recurrence and malignant progression of GBM.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0174. H3.3 K27M ACCELERATES PDGF-INDUCED BRAINSTEM GLIOMAGENESIS IN VIVO

Francisco Cordero 1, Peter Lewis 2, Christine Hoeman 1, Kristin Schroeder 1, C David Allis 2, Oren Becher 1

Abstract

INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brainstem tumor with a median survival time of less than one year. Recently, K27M H3.1 and H3.3 mutations were discovered to occur in 78% of DIPG patients and are indicative of a poor prognosis. This mutation nonsynonymously alters the specific, highly conserved lysine histone tail residue 27 to methionine which alters methylation patterns and subsequent gene expression. The goals of this study are to determine: 1) whether H3.3K27M is sufficient to induce brainstem gliomas, 2) if H3.3K27M accelerates PDGF-induced brainstem gliomagenesis, and 3) if H3.3K27M reduces global H3K27me3 levels in vivo. METHOD: We infected the brainstems of eight cohorts of nestin tv-a, p53 floxed mice with the following combinations of viruses: 1) H3.3K27M + Cre, 2) H3.3WT + Cre, 3) PDGF-B, 4) PDGF-B + H3.3K27M, 5) PDGF-B + H3.3WT, 6) PDGF + Cre, 7) H3.3K27M + PDGF + Cre, and 8)H3.3WT + PDGF + Cre. Mice were sacrificed upon developing tumor symptoms or at 12-weeks post infection. Mice without p53 loss were given an EdU injection 4hrs prior to sacrifice (200mg/kg). For all mice, brains were extracted, formalin fixed, and stained by immunohistochemistry or immunofluorescence for EdU, HA, PCNA, or H3K27me3. RESULTS: Expression of H3.3K27M with p53 loss in nestin progenitors induced small proliferating ectopic cell clusters in 72% of mice (n = 29). These clusters were not seen in mice with H3.3WT and p53 loss (n = 8). There was significantly increased proliferation with H3.3K27M; PDGF tumors relative to PDGF tumors or H3.3 WT; PDGF tumors as measured by EdU (8.0 ± 3.1 N = 5 vs. 2.2 ± 0.3 N = 6; p = 0.02 OR 8.0 ± 3.1 vs. 1.1 ± 0.1 N = 4; p = 0.02). The addition of H3.3K27M to PDGF; p53 deficient murine DIPGs significantly reduced global H3K27me3 (p < 0.0001). CONCLUSIONS: Expression of H3.3K27M with p53 loss in nestin expressing progenitors of the neonatal brainstem induced small ectopic proliferating cell clusters. These clusters may represent pre-neoplastic lesions. Surprisingly, H3.3K27M with p53 loss was not sufficient to induce gliomas. However, expression of H3.3K27M was shown to accelerate PDGF-induced brainstem gliomas by significantly increasing the proliferation rate. Furthermore, H3.3K27M is sufficient to reduce global H3K27me3 levels in vivo. We are currently investigating the mechanism by which H3.3K27M is cooperating with PDGF-induced brainstem gliomagenesis.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0175. A PHASE I/II STUDY OF ADOPTIVE T- CELL THERAPY (ALT) AND DC VACCINATION (DCV) DURING RECOVERY FROM MYELOABLATIVE CHEMOTHERAPY AND HEMATOPOIETIC STEM CELL TRANSPLANTATION (HDC + ASCR) OR NON-MYELOABLATIVE CONDITIONING (NMA) IN PATIENTS (PTS) WITH RECURRENT CENTRAL PNETS (RE-MATCH PROTOCOL)

Sridharan Gururangan 1, Gerald Grant 1, Tim Driscoll 1, Gerald Archer 1, James Herndon 1, Henry Friedman 1, Joanne Kurtzberg 1, Darell Bigner 1, John Sampson 1, Duane Mitchell 1

Abstract

INTRODUCTION: Outcome following standard therapy for children with recurrent central PNETs is extremely poor and novel therapies are required to improve outcome. We have developed a novel platform for the expansion of polyclonal populations of tumor-specific T cells using amplified tumor RNA pulsed DCs that has shown considerable efficacy in preclinical models of invasive brain cancers. We are conducting a phase I/II study to assess the feasibility, safety, and estimate the efficacy of ALT + DCV following recovery from HDC +ASCR (group A) or NMA (Group B) in patients with recurrent PNET (Re-MATCH Protocol, FDA IND BB-14058, Duke IRB #18020). METHOD: Eligible pts underwent surgical resection to confirm diagnosis and obtain tumor for vaccine preparation. Pts with localized recurrence (group A) or metastatic recurrence (group B) subsequently received 4 cycles of standard induction chemotherapy prior to either HDC + ASCR (carboplatin, thiotepa, and etoposide) (group A) or NMA using cyclophosphamide + fludarabine (group B). Three doses of ALT are evaluated (3 x (10^6, 10^7, 10^8) cells/Kg) with a single dose of intradermal DCV (107 cells). Safety evaluation for dose limiting toxicity (DLT) assessment is two weeks past the third biweekly vaccine with monthly vaccines thereafter as available. RESULTS: 19 subjects have been enrolled on a screening consent for collection of tumor tissue. Tumor RNA was successfully amplified from 13/13 recurrent tumor samples. Six samples were removed for histology other than PNET or no viable tumor on biopsy or resection. We have generated sufficient DCs for at least 3 vaccinations and T cells to the targeted dose in 12 of 13 enrolled subjects. Seven subjects have received immunotherapy; 4 subjects at 3 x 10^6 T cells/Kg (Group A-1, Group B-3), and 3 subjects in Group B at 3 x 10^7 T cells/Kg. There have been no DLTs observed. CONCLUSIONS: ALT + DCV therapy is feasible and preliminary evidence demonstrates the safety of this treatment approach. Accrual continues on the phase I component of the study along with clinical and immunologic evaluation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0176. NG2 UPREGULATION IN PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMA AND ITS ROLE IN TUMORIGENECITY IN VIVO

Sridevi Yadavilli 1, Madhuri Kambhampati 1, Oren Becher 2, Tobey MacDonald 3, Ravi Bellamkonds 4, Roger Packer 1, Javad Nazarian 1

Abstract

INTRODUCTION: Pediatric brainstem glioma (BSG) is one of the most difficult cancers to treat accounting for 10-20% of all pediatric central nervous system (CNS) tumors. BSGs may occur throughout the brainstem and are categorized into two main groups: diffuse intrinsic pontine gliomas (DIPGs) and focal brainstem gliomas. DIPGs represent about 80% of BSGs with a peak onset of six to nine years of age. DIPGs invade throughout the pons and may spread to other portions of brainstem. To better understand the pathophysiology of the disease, a genetically engineered (PDGFB-expressing) BSG mouse and a xenograft model have been established. METHOD: Protein from BSG mouse tumor and normal specimens were processed and submitted for MS/MS proteomic analysis using LTQ-Orbitrap-XL. Isolated peptides identified using the Sequest algorithm in the Bioworks browser against the Uniprot database were submitted to quantitative and subgroup analysis using ProteoIQ and Partek Genomics Suite. Proteins of interest were validated using human DIPG and mouse BSG specimens by western blot and immunohistochemical staining. In vivo assays were done using mouse and human DIPG primary cells. RESULTS: We show high expression of NG2 in murine model of brainstem glioma as well as 80% of pediatric DIPG specimens tested. shRNA-mediated knockdown of NG2 reduces cellular migration in vitro. NG2 expression is defective (symmetric) in mitotic cells in vitro and in vivo which is consistent with observations in adult gliomas. Injection of NG2 expressing neurospheres into brainstems of P2 mice results in highly aggressive brainstem tumors (leading to death within 3-7 weeks) providing a solid model for testing preclinical evaluations. Furthermore, we show selective delivery of liposomal nanoparticles to brainstem of our robust BSG mouse model. CONCLUSIONS: We introduce a robust murine model of brainstem glioma that is developed using NG2 expressing cells. High expression of NG2 in a subset of DIPGs and its defective expression may provide novel approaches for treating DIPGs and BSGs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0177. AN OPEN-LABEL, TWO-STAGE, PHASE II STUDY OF BEVACIZUMAB AND LAPATINIB IN CHILDREN WITH RECURRENT OR REFRACTORY EPENDYMOMA: A COLLABORATIVE EPENDYMOMA RESEARCH NETWORK STUDY (CERN)

Mariko DeWire 1, Maryam Fouladi 1, Clinton Stewart 2, Cynthia Wetmore 2, Cynthia Hawkins 3, Carmen Jacobs 4, Ying Yuan 4, Stewart Goldman 5, Paul Fisher 6, Michael Rytting 4, Eric Bouffet 3, Yasmin Khakoo 7, Eugene Hwang 8, Nicholas Foreman 9, Mark Gilbert 4, Richard Gilbertson 2, Amar Gajjar 2

Abstract

INTRODUCTION: Co-expression of ERBB2 and ERBB4 is reported in 75% of pediatric ependymomas and correlates with poorer overall survival. The maximum tolerated dose of lapatinib, a selective ERBB1 and ERBB2 inhibitor, in children with recurrent brain tumors is 900 mg/m2/dose BID, resulting in prolonged disease stabilization in ependymoma patients. We have demonstrated that endothelial cells interact with cancer stem-like cells in the vascular niche and bevacizumab exposure in ependymoma xenografts leads to ablation of tumor self-renewing cells, arresting growth. Based on these data, we conducted an open-label, phase II study of bevacizumab and lapatinib in children with recurrent or refractory ependymomas. METHOD: Patients were ≤ 21 years of age with recurrent or refractory intracranial or spinal ependymoma confirmed on central pathology review. Lapatinib was administered orally twice daily at 700 mg/m2/dose and bevacizumab given intravenously every two weeks at 10 mg/kg/dose. A course was defined as 28 days. Lapatinib trough levels were analyzed prior to each course in consenting patients. The expression of total and phosphorylated VEGFR2 were measured in peripheral blood mononuclear cells (PBMNCs) prior to the first and second doses of bevacizumab and 24-48 hours following the second dose of bevacizumab. RESULTS: Twenty-four patents were enrolled. Two were ineligible as tumor material was unavailable for centralized review. Among eligible patients, the median age was 10 years (range 2-21). Thirteen patients had anaplastic ependymoma; 20 were evaluable for response. There were no objective responses, but 4 patients had stable disease for ≥ 4 courses (range: 4-9). This did not reach stage I efficacy of 3/20 objective responses. Grade 3 toxicities included rash, hypokalemia, elevated ALT, and diarrhea. The only grade 4 toxicity was a peri-tracheostomy hemorrhage. Lapatinib plasma pharmacokinetics and phosphorylated and total VEGFR2 expression in PBMCs are currently being analyzed. CONCLUSIONS: Although the combination of bevacizumab and lapatinib was well-tolerated in children with recurrent ependymoma, it proved ineffective in this patient-population. Supported by CERN, Glaxo Smith Kline, Genentech

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0178. COMPARATIVE MOLECULAR ANALYSIS OF PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMA REVEALS TUMOR SUBTYPES WITH DIFFERENTIAL ACTIVITY OF SONIC HEDGEHOG PATHWAY

Amanda Saratsis 1, Sridevi Yadavilli 2, Kendall Snyder 3, Madhuri Kambhampati 2, Jordan Hall 4, Eric Raabe 5, Kathy Warren 6, Roger Packer 2, Javad Nazarian 2

Abstract

INTRODUCTION: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n = 16) and normal brain tissue (n = 10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. METHOD: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. RESULTS: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p<0.05, FC >2 or <-2). Unsupervised clustering revealed two distinct subgroups with differential ShH activity (GLI1 z-score -2.000 vs 2.000) and expression of GLI1, GLIPR1, PTCHD1 and SMO. Protein profiling revealed high expression of TLN1, CLU, EEF2 (FC >2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p<0.05, FC >2 or <-2). Concordant differences in DNA methylation were detected in related genes, including GLI1, FOXF1, SMO, SHH, and SUFU (ANOVA, p < 0.05, FC<-2 or >2). CONCLUSIONS: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0179. CORRELATION OF CSF AND SERUM BIOMARKERS OF OXIDATION IN PEDIATRIC BRAIN TUMOR PATIENTS

Joshua Thompson 1, Andrea Griesinger 1, Nicholas Foreman 1, Sarah Rush 2, Jean Mulcahy Levy 1

Abstract

INTRODUCTION: There is interest in identifying biological markers (biomarkers) that can be measured for diagnosis, prognosis, and evaluating response to therapies. Increases in measures of oxidative stress in CSF have been shown to be an indicator of brain injury and these could be useful in our patient population, although measuring these markers in the CSF poses an obstacle as CSF is not routinely collected during therapy. In order to use markers of oxidative stress, a less invasive method of monitoring is needed. We hypothesized blood markers of oxidative stress would correlate with CSF levels providing a non-invasive method of monitoring. METHOD: One measure of oxidative stress is determined by the concentration of F2 isoprostanes, a prostaglandin-like compound formed from peroxidation of arachidonic acid present in high concentration in phospholipids. We collected matched pairs of CSF and serum from patients at the time of primary tumor resection. F2 isoprostane levels were measured in each sample by a competitive enzyme-linked immunoassay (ELISA) (Cayman's 8-Isoprostane EIA Kit). Correlation between the CSF and serum values was determined using a Pearson's correlation. RESULTS: Reproducible and reliable values were able to be obtained from both the serum and CSF samples from patients with multiple tumor types. We also found a strong correlation of F2 isoprostane between CSF and serum with an R-value of 0.75. CONCLUSIONS: This study demonstrates a correlation between serum and CSF levels of F2 isoprostane, a marker that has been previously shown to correlate with levels of oxidative brain injury. This study will allow for further investigations into the prognostic applicability of oxidative markers in children with CNS tumors as our study has shown that serum levels correspond with those in CSF and provide a less invasive mechanism for study. Future studies are needed to determine a correlation between the levels of these oxidative markers in the serum and the long-term outcomes in children with CNS tumors.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0180. mTORC1/mTORC2 SIGNALING IN PILOCYTIC ASTROCYTOMA

Marianne Hutt 1, Matthias A Karajannis 2, Smit Shah 1, Charles G Eberhart 1, Eric Raabe 1, Fausto J Rodriguez 1

Abstract

INTRODUCTION: Previous studies support a role for MAPK pathway signaling, and more recently AKT/mTOR, in pilocytic astrocytoma biology. mTOR operates as part of two different multiprotein signaling complexes, mTORC1 and mTORC2. In the current study we evaluated mTORC1/mTORC2 pathway activity in pilocytic astrocytoma. METHOD: We used formalin-fixed paraffin embedded human pilocytic astrocytomas with immunohistochemistry in tissue microarrays. We also studied in vitro the effect of mTORC1 blockade in pediatric low grade glioma cell lines Res 186 (derived from a pilocytic astrocytoma) and Res 259 (derived from a diffuse astrocytoma) with a TORC1 inhibitor. Cell growth was assessed with the MTT assay. RESULTS: Moderate(2+) to strong(3+) immunostaining was observed for pS6 66/114(58%), p4EBP1 35/116(30%), pElF4G 66/113 (58%), mTOR(total) 52/114 (46%), Raptor 64/102(63%), Rictor 48/101(48%), and pAKT(S473) 63/103(61%). Complete PTEN protein loss was identified in 7/101 (7%). There was increased immunoreactivity in pilocytic astrocytomas of the optic pathways compared with other sites for pS6, pElF4G, mTOR(total), Rictor, and pAKT (p < 0.05, Wilcoxon Rank Sum), but not for p4EBP1(p = 0.28), Raptor(p = 0.15) or PTEN(p = 0.68). Cell growth was inhibited (Res 186 > Res 259) with 1nM and 10nM drug concentrations, compared with DMSO (p < 0.005, t-test). Additionally, pS6 levels were decreased by Western Blot in Res 186 cells, but not in Res 259. CONCLUSIONS: The findings support that the mTOR pathway is active in a subset of pilocytic astrocytoma/pediatric low grade glioma, and varies by clinicopathologic subtype. Additionally, our data suggests that mTORC2 is differentially active in optic pathway gliomas, but not mediated by PTEN loss. Furthermore, mTOR may represent a therapeutic target in pediatric low grade glioma that merits further investigation.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0181. IDENTIFICATION OF AREG, SHH, MMP12 AND MMP9 AS NOVEL THERAPEUTIC TARGETS IN CRANIOPHARYNGIOMA

Jacob Gump 1, Andrew Donson 1, Diane Birks 1, Michael Handler 2, Nicholas Foreman 2, Todd Hankinson 2

Abstract

INTRODUCTION: Neurological consequences of craniopharyngioma (CPA) and its treatment condemn children with this disease to the lowest overall quality of life of any primary pediatric brain tumor population. This is due to irreversible neurological damage, including hypothalamic obesity, panhypopituitarism, and blindness. However, CPA is woefully understudied due to its low mortality (>90% 5-year survival), clinical complexity and recalcitrance to laboratory study. CPA is the most common non-glial pediatric brain tumor (6-8%). Yet, there are no proven pharmacologic therapies for CPA, limiting treatment to surgery and radiation. Here, we identify rational targets for CPA therapy. METHOD: This pilot study employed gene expression microarray analysis to study 8 samples of snap-frozen pediatric CPA to identify genes that were substantially overexpressed relative to normal human brain, normal pituitary and a panel of pediatric brain tumors. Overexpression was confirmed by western blotting for protein levels and by quantitative RT-PCR. We also analyzed each sample for activating mutations in B-catenin by sequencing the hot-spot region in exon 3. Clustering analyses were also conducted using microarray data from CPA and other CNS tumors to reveal gene expression patterns and signatures in the CPA samples. RESULTS: We identified the overexpression of 4 targetable gene products in this panel of CPA samples. Amphiregulin (AREG) mRNA was overexpressed by an average of 37-fold above normal brain tissue and other pediatric brain tumors. We also identified 162-fold overexpression of sonic hedgehog (SHH). Furthermore, CPA specimens expressed matrix metalloprotease-12 (MMP-12) and MMP-9 at levels 1400 times and 900 times higher, respectively, than any other tumor tested. The overexpression of these genes was confirmed by QPCR and using western blot analysis, which demonstrated that these targets were expressed exclusively in CPA and not in normal brain or any pediatric CNS tumor. CONCLUSIONS: Each of these molecules represents a rational therapeutic target for CPA. Medications targeting these molecules are in or approaching clinical trials but have not yet been tested against CPA. These preliminary findings will be confirmed in a larger cohort of CPA samples, with the goal of providing robust biological data that can drive subsequent clinical trials. Treatments and outcomes for CPA have remained essentially unchanged for decades; targeted therapy has the potential to change this paradigm.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0182. INTEGRATIVE GENOMIC ANALYSES IDENTIFY RECURRENT STRUCTURAL ALTERATIONS IN ATYPICAL TERATOID RHABDOID TUMOURS (ATRTS)

Jonathon Torchia 1, Dong-Anh Khuong-Quang 2, King Ching Ho 1, Daniel Picard 1, Louis Letourneau 3, Tiffany Chan 1, Brian Golbourn 1, Sorana Morrissy 4, Diane Birks 5, Claudia Faria 4, Nick Foreman 7, Michael Taylor 4, James Rutka 4, Stefan Pfister 6, Eric Bouffet 4, Cynthia Hawkins 4, Jacek Majewski 2, Seung-Ki Kim 8, Nada Jabado 2, Annie Huang 2

Abstract

INTRODUCTION: ATRTs (Atypical teratoid rhabdoid tumours) represent one of the most aggressive pediatric brain tumours. However, paradoxically, ATRTs have been reported to exhibit balanced genomes with only recurrent somatic alterations of the SMARCB1 locus on chr22. To better define molecular mechanisms underlying ATRT biology we comprehensively interrogated 63 ATRTs using an integrated genomics approach. METHOD: 63 ATRTs were investigated using a combination of OmniQuad ultra-high resolution SNP genotyping (n = 39) and whole-genome/exome sequence (n = 42) analyses. Copy number and structural alterations were mapped using orthogonal methods including circular binary segmentation (dChip, Partek Suites), CREST, BreakDancer and Pindel analyses. Alterations were validated by targeted re-sequencing using the Sanger method and/or MiSeq and Ion Torrent analyses. RESULTS: ATRTs exhibited few recurrent deleterious SNVs with exception of loss of function mutations in SMARCB1 (15 SNVs in 63 tumours). Significantly, ATRTs exhibited a predominance of structural events (∼3.13/tumour) including recurrent losses of LRP1B (chr2q22.1), CDH13 (chr16q23.3), BCR (chr22q11.21), PIWIL3 (chr22q11.23), CHEK2 (chr22q12.1), MKL1 and EP300 (chr22q13.12). We also detected inter-chromosomal translocations of EARS2/TRAM1 (t[8;9][q13;16p12]) and CDH13 with a non-coding locus (t[16;11][ q23;p15]). Notably a majority of ATRTs (48/63) exhibited structural alterations of SMARCB1 including 2 novel intra-chromosomal events identified by WGS that were not detectable by MLPA or exon sequence analyses. CONCLUSIONS: Somatic structural alterations drive recurrent genetic events in ATRT. Our integrated high resolution genomics approach has uncovered novel loci with predicted functions in cell adhesion, DNA damage response and epigenetic regulation that will inform a better understanding of ATRT tumour biology. The identification of novel structural events in SMARCB1 and other genes indicates that the scope of genetic alterations in ATRTs has to date been underestimated and underscore WGS as an important tool for gene discovery as well as clinical diagnostics in ATRT.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0183. AVOIDING CEREBELLAR MUTISM VIA THE TELOVELAR APPROACH TO POSTERIOR FOSSA TUMOR RESECTION IN CHILDREN

Travis Ladner 1, Luke Tomycz 1, Jennifer Watchmaker 1, Tiffany Yang 1, Laura Kaufman 1, Matthew Pearson 1

Abstract

INTRODUCTION: Cerebellar mutism is a common complication of posterior fossa surgery in the pediatric population, characterized by its delayed onset and limited duration of expressive aphasia or dysarthria. Bilateral lesioning of the dentate nuclei and their connections, as well as the vermis, during surgery is thought to account for this phenomenon. The telovelar approach provides access to the 4th ventricle through the cerebromedullary fissure, sparing unintended damage to midline structures. We present our experience with cerebellar mutism in 90 cases of posterior fossa tumors removed via the telovelar approach. METHOD: In the telovelar approach, a suboccipital craniotomy is made and the cerebromedullary fissue is carefully dissected and split into two spaces. The tela choroidea and inferior medullar make up the floor of the fissure, and once incised provide access to the fourth ventricle without splitting the vermis. A retrospective review of 90 consecutive pediatric patients undergoing resection of posterior fossa tumors via the telovelar approach between 2003 and 2013 was conducted. Patient demographic data, tumor type, size, and location, as well as presence of postoperative mutism, were assessed. RESULTS: 90 patients underwent excision of posterior fossa tumors via the telovelar approach. The mean patient age was 7.1 years. The most common tumors were pilocytic astrocytoma (37/90, 41.1%), medulloblastoma (28/90, 31.1%), and ependymoma (13/90, 14.4%). The mean tumor size was 4.2 cm. The mean follow up period was 35 months. 7/90 patients (7.8%) developed cerebellar mutism. The median latency and duration, respectively, were 4.5 and 19.5 days. In all cases of mutism, the tumor was in the midline. Medulloblastoma accounted for 3/7 cases, pilocytic astrocytoma for 3/7, and glioblastoma multiforme for 1/7. 12/90 patients were deceased at follow up. CONCLUSIONS: The traditional surgical approach to posterior fossa tumors involves a suboccipital craniotomy and splitting of the vermis, with lateral retraction of the cerebellar hemispheres. The telovelar approach obviates the need for vermis dissection and has been described previously with limited success in avoiding cerebellar mutism. Our experience reveals one of the lowest incidences of cerebellar mutism reported, showing a distinct advantage of the telovelar approach. We propose that careful dissection minimizes risk to nearby cerebellar structures, reducing the development of cerebellar mutism.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0184. NEURAL SYSTEMS FOR READING IN MEDULLOBLASTOMA SURVIVORS

Robert J Ogg 1, Matthew A Scoggins 1, Ping Zou 1, Samina Taherbhoy 1, Melissa M Jones 1, Yimei Li 1, John O Glass 1, Thomas E Merchant 1, Wilburn E Reddick 1, Heather M Conklin 1, Amar Gajjar 1

Abstract

INTRODUCTION: Children treated for medulloblastoma are vulnerable to a spectrum of cognitive deficits, including difficulty learning to read. Visual areas of the ventral occipital and temporal lobes play an important role in normal fluent reading. Based on converging neuropsychological, structural imaging, and functional imaging evidence from previous studies, we hypothesized that reading deficits are associated with therapy-induced disruption of ventral visual specialization during the development of reading skills. We used advanced functional neuroimaging methods to investigate in medulloblastoma survivors the relations between acquired reading deficits and neural systems for reading. METHOD: Participants gave informed consent for this IRB-approved study, and included 57 medulloblastoma patients (14.8 [6 – 27] year) and 97 healthy controls (13.8 [8 – 25] year). Patients were > 1 year (3.3 [1.8 – 6.1] year) after SJMB03 protocol therapy http://clinicaltrials.gov/ct2/show/study/NCT00085202). Longitudinal evaluations (baseline, +1 year, +2 year) included comprehensive neuropsychological testing and neuroimaging. Imaging included a battery of five fMRI tasks to probe ventral visual function in reading, diffusion tensor imaging to characterize white matter, and high-resolution structural MRI. Task activation (SPM, http://www.fil.ion.ucl.ac.uk/spm/) and connectivity (GIFT, http://mialab.mrn.org/software/gift/) were analyzed with clinical and behavioral covariates. RESULTS: Reading performance was diminished in patients (eg, reading fluency: patients = 93.3 [62 – 130], controls = 111.6 [82 − 175]) and patterns of brain activation during orthographic processing were altered in children who survive medulloblastoma. Activation was altered in neocortical networks for reading, and changes in activation were associated with performance on standardized tests of reading performance. Functional connectivity during orthographic processing revealed remarkable interactions among cortical and subcortical networks, including thalamus, basal ganglia, cerebellum, and brainstem. CONCLUSIONS: Functional neuroimaging demonstrated complex interaction between development and adverse brain function changes in children treated for medulloblastoma. Ventral visual processing was disrupted in patients, consistent with high radiation doses to cortex and commissural white matter connections around the posterior fossa. However, activation was affected broadly in patients, suggesting that disease and treatment lead to widespread brain changes. Connectivity mapping is an important complement to conventional structural and functional imaging, and promises to advance understanding of effects of disease and treatment on organization of brain networks, neural substrates of subsequent cognitive deficits, and the impact of interventions on brain function.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0185. PROMOTER METHYLATION-DEPENDENT SILENCING OF CELL CYCLE TUMOR SUPPRESSORS IN AGGRESSIVE PNET IS OVERCOME BY PERIFOSINE AND HDAC INHIBITORS

Asadullah Khan 1,2, Anil Kumar 4, Gary W Tye 2, William C Broaddus 2, Timothy E Van Meter 1,3

Abstract

INTRODUCTION: Primitive neuroectodermal tumors (PNETs) are frequently found to have amplifications of Cyclin D and Cyclin dependent kinase genes, which are major driving factors for the high mitotic rate and aggressive growth of these tumors. Previously our labs have shown that perifosine treatment of medulloblastoma and supratentorial PNET cultures and orthotopic xenografts induces dramatic p21-dependent growth arrest in PNET tumor models. To better understand the mechanism of induction of p21 following treatment, studies were performed to better characterize the process of transcriptional activation of p21 and the related cell cycle inhibitor p16. METHOD: Establishment of new cell and xenograft models from sPNET in our laboratories address the need for more appropriate preclinical models. Primary tumor tissue, short term PNET cultures and their xenografts were examined for P21 and P16 expression by quantitative RT-PCR prior to and following treatment for 24 hours with perifosine, CUDC101, SAHA and valproic acid, compared to non-tumor CNS stem cell cultures and normal brain samples. Extracts were subjected to bisulfite sequencing and the results compared. Changes in expression levels of P21,P16 and cell lineage and differentiation markers were assayed by Taqman qRT-PCR assay, confocal imaging and western blotting. RESULTS: Bisulfite sequencing revealed that both genes were methylated at cytosine residues within and outside of typical CpG islands. A similar reversal of cytosine methylation was observed after treatment with histone deacetylase (HDAC) inhibitors SAHA, CUDC101 and valproic acid. The reactivation of p16 and p21 in multiple tumor models was sufficient to overcome noted amplifications in the Cyclin/CDK pathway and produced significant attenuation of tumor growth, leading an increase in expression of terminal differentiation markers. CONCLUSIONS: These data indicate that treatment of sPNET with the multikinase inhibitor perifosine, or with HDAC inhibitors already in clinical trials may be dependent upon reversal of DNA methylation-mediated silencing of p21 and/or p16, and provide a way of assaying the therapeutic response of treated tumors within future clinical trial designs.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0186. COMBINED CLINICAL AND CYTOGENETIC PROGNOSTIC MARKERS GREATLY ENHANCES RISK-STRATIFICATION OF MEDULLOBLASTOMA

David J H Shih 2, Paul A Northcott 3, Marc Remke 1, Andrey Korshunov 4, David T W Jones 3, Marcel Kool 3, Stefan M Pfister 5, Michael D Taylor 6; Medulloblastoma Advanced Genomics International Consortium1

Abstract

INTRODUCTION: Medulloblastoma is the most common malignant pediatric brain tumour, and it comprises four molecular subgroups - WNT, SHH, Group3, and Goup4 - which have disparate biological, clinical, and demographic characteristics. Current treatment regimens stratify all medulloblastoma patients into risk groups based on clinical features including patient age, tumour histology, and metastatic stage. Recent reports of the stark genetic differences among the four subgroups of medulloblastoma suggest that subgroup-specific biomarkers may improve the risk-stratification of patients. METHOD: Molecular biomarkers for risk-stratification were identified from a copy-number profiling screen by single nucleotide polymorphism arrays on a discovery set (n = 629), consisting of samples acquired retrospectively from 43 cities across the globe. Tissue microarray analyses for select biomarkers were performed in an independent validation set (n = 453). Combined risk-stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariate Cox proportional hazard analyses on the discovery set, and the models were subsequently tested on the validation set. RESULTS: Subgroup status was strongly predictive of patient survival; it improved the predictive accuracy of a multivariate survival model on top of known clinical biomarkers. WNT patients had good survival irrespective of clinical biomarkers. With the exception of metastatic stage, most clinical biomarkers were differentially associated with survival across the remaining subgroups. Similarly, most known cytogenetic biomarkers were only prognostic within specific subgroups (e.g. MYC and MYCN amplifications). Our clinical-cytogenetic risk schema can identify low-risk (almost Wnt-like) Group3 patients, and high-risk (almost Group3-like) SHH patients using only tools available in a modern neuropathology laboratory. CONCLUSIONS: Molecular subgroups and genomic aberrations in medulloblastoma are powerful biomarkers of prognostic outcome. Combining subgroup and cytogenetic biomarkers with established clinical risk factors can substantially improve survival prediction, even in the context of heterogeneous clinical treatments. Critically, the prognostic significances of biomarkers are often restricted to specific subgroups. By examining >1000 patients, we have identified several novel cytogenetic markers that identify very high risk, and very low risk groups of patients. The identified cytogenetic biomarkers can serve as high-priority candidates for prospective multicentre trials.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0187. SONIC HEDGEHOG SIGNALING AND MEDULLOBLASTOMA PROGRESSION

Frederic Mille 1, Martin Levesque 1, Marc Remke 3, Andrey Korshunov 3, Luisa Izzi 1, Marcel Kool 3, Paul A Northcott 2, Michael D Taylor 2, Stefan M Pfister 3, Frederic Charron 1

Abstract

INTRODUCTION: During cerebellar development, the Sonic hedgehog (Shh) signaling pathway drives the proliferation of granule cell precursors (GCPs). Aberrant activation of Shh signaling causes over-proliferation of GCPs leading to medulloblastoma, an aggressive pediatric brain tumor. Although the tissue-restricted Shh-binding protein Boc associates with the Shh receptor Ptch1 to mediate Shh signaling, whether Boc plays a role in medulloblastoma tumorigenesis is unknown. METHOD: n/a RESULTS: Here we show that BOC is upregulated in the SHH subgroup of human medulloblastomas and that Boc upregulation is associated with the early steps of medulloblastoma tumorigenesis in a mouse model of medulloblastoma. We further show that Boc upregulation in GCPs induces their proliferation. Conversely, Boc inactivation decreases the size of medulloblastoma tumors and reduces the progression of early medulloblastomas into advanced tumors. We find that Boc regulates tumor size by controlling the expression of genes important for cell proliferation. Remarkably, Boc also regulates Ptch1 loss-of-heterozygosity, an important event in the progression from early to advanced medulloblastoma. CONCLUSIONS: Taken together, our results indicate that Boc promotes medulloblastoma progression and that targeting tissue-restricted components of the Shh signaling pathway, such as Boc, can prevent cancer development.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0188. HIGH-THROUGHPUT CHEMICAL BIOLOGY SCREEN IDENTIFIES ROS INTOLERANCE AS A VULNERABILITY OF MYC-AMPLIFIED/GROUP 3 MEDULLOBLASTOMAS

Furong Yu 1, Sabran Masoud 1, Brian Nguyen 1, Nusi Vue 1, Simone Schubert 1, Nicola Tolliday 2, Soma Sengupta 3, Dilhan Weeraratne 3, Stuart Schreiber 2, Yoon-Jae Cho 1

Abstract

INTRODUCTION: Poor outcome in medulloblastoma is driven in part by MYC-amplified / Group 3 medulloblastomas. These tumors are notably resistant to standard chemotherapy and radiotherapy, even at maximally tolerated doses. Understanding which molecular pathways, when targeted, result in a biologically significant effect will help guide the development of novel therapies for children with these lethal tumors. METHOD: We performed a luciferase-based cell viability screen on a patient-derived MYC-amplified medulloblastoma cell line against a library of 1,982 biologically and structurally diverse compounds. We identified 115 “hits” from the primary screen and performed secondary validation of these compounds against four additional MYC-amplified medulloblastoma cell lines and a counter screen in normal subventricular zone derived neural stem cells. Chemical genomic signatures of selected candidates were generated and gene set enrichment analysis was performed against databases containing canonical pathways and chemical genomic signatures of other well-annotated drugs. Further confirmation of inferred biological activity was performed using standard bench assays. RESULTS: We identified several classes of compounds that effectively inhibited MYC-associated transcriptional activity and decreased viability in Group 3 medulloblastoma cells. These included proteasome inhibitors, aldehyde dehydrogenase inhibitors, HDAC inhibitors and several neuroactive ligand receptor agonists. Despite the variation of mechanism of action across these compounds, the overlaps of their gene expression profiles identified enrichment of genes corresponding to cellular response to ROS. Increased ROS was confirmed by DCFDA staining and inhibition of their biological effects by co-treatment with NAC, a ROS scavenger. CONCLUSIONS: Using a chemical biology and chemical genomics approach, we have identified several classes of compounds that shut down MYC transcriptional activity and effectively inhibit MYC-amplified medulloblastoma growth and survival. Though these individual classes of compounds target different proteins/pathways, they all converge on cellular response to ROS. Our approach highlights various mechanisms, including proteasome inhibition and inhibition of aldehyde dehydrogenase activity, by which increases in ROS can be achieved and such agents should be considered for further preclinical and clinical development.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0189. HIGH THROUGHPUT SEQUENCING REVEALS SMARCB1 LOSS RESULTS IN STABLE GENOME BUT VARIABLE TRANSCRIPTOME IN AT/RTS

Diane Birks 1, Ken Jones 1, Andrea Griesinger 1, Vladimir Amani 1, Michael Handler 2, Rajeev Vibhakar 1, Nicholas Foreman 1

Abstract

INTRODUCTION: AT/RTs are aggressive early childhood brain tumors that are characterized by the functional loss of SMARCB1 through biallelic deletion and/or mutation. A recent study using Whole Exome Sequencing of DNA (WES) identified an extremely low mutation rate in AT/RTs, and no genes altered recurrently other than SMARCB1 (Lee et al, 2012). These findings suggest that factors other than genomic instability are important drivers of AT/RTs. Here we report the results of both WES and high-throughput RNA sequencing (RNA-seq) in a matched cohort of AT/RTs to extend these genomic findings and to look for alterations in the AT/RT transcriptome. METHOD: DNA was extracted from 5 AT/RT patient samples and 5 matched germline samples and used for WES on the Illumina platform. Reads were aligned using the Burrows-Wheeler-Alignment algorithm, and variants were called using the Genome Analysis Tool Kit. Whole transcriptome sequencing of polyA+ RNAs (RNA-seq) was performed on the Illumina platform using 4 of the tumors and 2 of the germline samples used for WES. The RNA-seq reads were aligned using GSNAP, and differential expression of genes and isoforms was examined using Cufflinks tools, DESeq and DEXSeq. RNA-seq reads were also used to verify genomic alterations identifed by WES. RESULTS: WES resulted in a mean coverage of 77-fold over the targeted genome of 51.6 Mb, with over 80% of sites achieving "callable" coverage of 10X or greater. Mean mutation rate was .21/Mb. The only gene altered in more than one tumor sample was SMARCB1. RNA-seq resulted in an average of 74M read-pairs/sample, with 84% alignable. RNA-seq levels correlated highly to gene expression measured by microarray for the same samples (>.75). RNA expression levels showed a high level of heterogeneity between the AT/RT samples. Differential exon usage in AT/RTs compared to normals was seen in 1% of genes. CONCLUSIONS: The DNA sequencing results closely matched those reported previously by Lee et al (2012). In spite of the very low level of genomic alterations in these tumors, RNA-sequencing revealed substantial heterogeneity across the AT/RT transcriptomes, both at the gene and isoform levels. The results of this study suggest that loss of SMARCB1 does not result in genomic instability, but does impact the transcriptome in ways that are often occurence-specific. The reason(s) underlying this heterogenity remain to be discovered.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0190. CHARACTERIZATION OF RADIATION-INDUCED BRAIN INJURY IN SURVIVORS OF PEDIATRIC BRAIN TUMORS USING MAGNETIC RESONANCE SPECTROSCOPY

RJ Brown 1, K Rangan 2, JL Finlay 1, A Olch 3, D Freyer 2, S Bluml 4

Abstract

INTRODUCTION: Children receiving radiation therapy for malignant brain tumors are at risk for developing long-term sequelae including endocrinopathies, cognitive deficits, and secondary malignancies. These deficits take years to develop hampering the ability to conduct efficient clinical trials with toxicity endpoints and develop interventions to mitigate these effects. Magnetic resonance spectroscopy (MRS) is a non-invasive imaging technique that can characterize a variety of brain injuries. We explore the MRS characteristics of patients with radiation-induced brain injury. METHOD: Survivors of pediatric brain tumors who received cranial irradiation and underwent clinical MRS of either frontal or parietal white matter using a standard single voxel PRESS sequence during routine MR follow-up were included. Comparisons were made with anonymized control data. Means and standard deviations were calculated and 2-sided Student t-tests were performed for 15 measures. Bonferroni correction was used for multiple comparisons. RESULTS: 10 patients were identified with 14 white matter spectra. Patients received cranial irradiation of 18Gray (n = 1), 23.4Gy (n = 3), 30.6Gy (n = 1), or 36Gy (n = 5) with a boost to the posterior fossa (n = 9) or pineal region (n = 1). Median age at time of irradiation was 5.8 years (range: 4.3, 18). Median time between irradiation and MRS was 12.3 years (range: 1.7, 17.9); and median age at time of MRS was 18.8 years (range: 10.5, 25.9). 35 control spectra from 4 control patients with a median age of 27.2 years (range: 26.6, 40.9) were used. Of the metabolites reviewed, choline:creatine ratios were statistically significantly greater in survivors when compared to controls (p < 0.001). CONCLUSIONS: Although the number of spectra was small in this very heterogeneous cross-sectional cohort, there appear to be significant differences in the metabolic profiles which may correlate with the pathogenesis of radiation-induced brain injury. Elevated choline:creatine ratios, for example, have been observed in neuroinflammatory conditions. Studies with more patients are currently ongoing.

Neuro Oncol. 2013 Apr;15(Suppl 1):i1–i51.

0191. ABROGATION OF T CELL TGF-Β SIGNALING RESTRICTS MEDULLOBLASTOMA PROGRESSION

David Gate 1, Moise Danielpour 2, Javier Rodriguez 1, Gi Bum Kim 1, Rachelle Levy 1, Serguei Bannykh 3, Joshua J Breunig 1, Terrence Town 4

Abstract

INTRODUCTION: Transforming growth factor beta (TGF-β) is a pleiotropic cytokine regulator of the immune system, and is required for development of suppressive T regulatory cells (Tregs). Immunosuppression via the secretion of TGF-β is a potent mechanism for human cancers to evade the immune response. We sought to determine the impact of genetically ablating TGF-β T cell signaling on progression of medulloblastoma, a highly malignant pediatric brain tumor that is the most common childhood central nervous system cancer. METHOD: A mouse model expressing a dominant-negative form of the TGF-β receptor type II under T cell regulatory control (TGFbRII-DNR) was utilized to inhibit T cell TGF-β signaling. This transgenic expresses a type II TGF-β receptor that lacks the intracellular kinase domain and acts as a dead-end decoy receptor to bind TGF-β ligand at the cell membrane. These mice were bred with mice carrying a mutant smoothened transgene (SmoA1). These animals have constitutively active Sonic hedgehog signaling in cerebellum and develop medulloblastomas. RESULTS: CD8+ cytotoxic T lymphocytes (CTLs) were found in TGFbRII-DNR+/SmoA1+ medulloblastomas and were nearly absent in TGFbRII-DNR−/SmoA1+ control tumors. These CTLs conferred anti-tumor immunity, operationalized as ∼35% increased survival of TGFbRII-DNR+/SmoA1+ vs. TGFbRII-DNR−/SmoA1+ mice. Finally, CTL anti-tumor immunity could be propagated from one animal to another in adoptive transfer experiments. CONCLUSIONS: Because TGFbRII-DNR mice lack functional Tregs, these results raise the possibility of a translational approach to medulloblastoma immunity by blocking TGF-β signaling and thereby depleting Tregs that promote tumor growth.

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

RESOURCES